MX2007007854A - Novel variant hypocrea jecorina cbh2 cellulases. - Google Patents

Abstract

Described herein are variants of <i>H. jecorina</i> CBH2, a Cel6A enzyme. The present invention provides novel cellobiohydrolases that have altered thermostability.

Description

NEW VARIANTS OF CELLULASES CBH2 OF HYPOCREA JECORINA FIELD OF THE INVENTION The present invention relates to cellobiohydrolase variant enzymes and isolated nucleic acid sequences encoding polypeptides having cellobiohydrolase activity. The invention also relates to nucleic acid constructs, vectors and host cells comprising the nucleic acid sequences, as well as methods for producing the recombinant, variant CBH polypeptides. BAOUND OF THE INVENTION Cellulose and hemicellulose are the most abundant plant materials produced by photosynthesis. These can be degraded and used as a source of energy by numerous microorganisms, including bacteria, yeasts and fungi, which produce extracellular enzymes capable of hydrolysis of the polymeric substrates to monomeric sugars (Aro et al., J. Biol. Chem. , Vol. 276, No. 26, pp. 24309-24314, June 29, 2001). As the limits of non-renewable resources approach, the potential of cellulose to become a major source of renewable energy is enormous. Krishna et al., Bioresource Tech. 77: 193-196, 2001). The effective utilization of cellulose through biological processes is a REF procedure. : 182789 to overcome the shortage of food, food and fuel (Ohmiya et al., Biotechnol.Gen Engineer, Rev. Vol. 14, p.365-414, 1997). Cellulases are enzymes that hydrolyze cellulose (beta-1, 4-glucan or beta-D-glucosidic bonds) resulting in the formation of glucose, cellobiose, cellooligosaccharides, and the like. Cellulases have traditionally been divided into three main classes: endoglucanases (EC 3.2.1.4) ("EG"), exoglucanases or cellobiohydrolases (EC 3.2.1.91) ("CBH") and beta-glucosidases ([beta] -D-glucoside -glucohydrolase; EC 3.2.1.21) ("BG"). (Knowles et al., TIBTECH 5, 255-261, 1987; Schülein, Methods Enzymol., 160, 25, pp. 234-243, 1988). The endoglucanases act mainly on the amorphous parts of the cellulose fiber, while the cellobiohydrolases are also capable of degrading the crystalline cellulose (Nevalainen and Penttilá, Mycota, 303-319, 1995). Thus, the presence of a cellobiohydrolase in a cellulase system is required for the efficient solubilization of crystalline cellulose (Suumakki, et al., Cellulose 7: 189-209, 2000). Beta-glucosidase acts to release D-glucose units from cellobiose, cello-oligosaccharides, and other glycosides (Freer, J. Biol. Chem. Vol. 268, No. 13, p.9337-9342, 1993). It is known that cellulases are produced by a large number of bacteria, yeasts and fungi. Certain mushrooms they produce a complete cellulase system capable of degrading the crystalline forms of cellulose, such that cellulases are easily produced in large quantities by means of fermentation. Filamentous fungi play a special role since many yeasts, such as Sa ccharomyces cerevisiae, lack the ability to hydrolyze cellulose. See, for example, Aro et al., 2001; Biochemistry and Genetics of Cellulose Degradation, eds. Aubert, J. P. et al., Academic Press, 1988; Ood et al., Methods in Enzymology, Vol. 160, No. 9, p. 87-116, 1988, and Coughlan, et al. "Comparative Biochemistry of Fungal and Bacterial Cellulolytic Enzyme Systems" Biochemistry and Genetics of Cellulose Degradation, pp. 11-30 1988. The fungal cellulase classifications of CBH, EG and BG can also be expanded to include multiple components within each classification. For example, multiple CBHs, EGs and BGs have been isolated from a variety of fungal sources including Tri choderma reesei (also referred to as Hypocrea j ecorina) which contains known genes for 2 CHBHs, eg, CBH I ("CBH1") and CBH II ("CBH2"), at least 8 EGs, eg EG I, EG II, EG III, EGIV, EGV, EGVI, EGVII and EGVIII, and at least 5 BGs, eg, BG1, BG2, BG3, BG4 and BG5. EGIV, EGVI and EGVIII also have xyloglucanase activity.

In order to efficiently convert crystalline cellulose to glucose, the cellulase system comprises the components of each of the classifications of CBH, EG and BG is required, with the isolated components less effective in hydrolyzing crystalline cellulose (Filho et al. , Can. J. Microbiol. 42: 1-5, 1996). A synergistic relationship between the cellulase components of different classifications has been observed. In particular, EG cellulases and CBH cellulases interact synergistically to more efficiently degrade cellulose. See, for example, Wood, Biochemical Society Transactions, 611th Meeting, Galway, Vol. 13, p. 407-410, 1985. Cellulases are known in the art to be useful in the treatment of textile materials for purposes of improving the cleansing ability of detergent compositions, for use as a softening agent, to improve the feel and appearance of cotton fabrics, and the like (Kumar et al., Textile Chemist and Colorist, 29: 37-42, 1997). The cellulase-containing detergent compositions with improved cleaning performance (U.S. Patent No. 4,435,307; U.S. Patent Applications Nos. 2,095,275 and 2,094,826) and for use in the treatment of fabric to improve the feel and feel of textile materials (Patents of the United States Nos. (US Pat Nos. 5,648,263, 5,691,178, and 5,776,757; British Patent Application No. 1,358,599; The Shizuoka Prefectural Hamamatsu Textile Industrial Research Institute Report, Vol 24, pp. 54-61, 1986), have also been described. Therefore, cellulases produced in fungi and bacteria have received significant attention. In particular, it has been shown that the fermentation of Trichoderma spp. (for example, Tri choderma longibrachia tum or Trichoderma reesei) produces a complete cellulase system capable of degrading the crystalline forms of cellulose. Although cellulase compositions have been previously described, there remains a need for new and improved cellulase compositions for use in household detergents, stone washing compositions or laundry detergents, etc. Cellulases that show improved performance are of particular interest. BRIEF DESCRIPTION OF THE INVENTION The invention provides an isolated cellulase protein, identified herein as variant CBH2, and the nucleic acids encoding a variant of CBH2. In one embodiment, the invention is directed to a variant cellulase of CBH2, wherein the variant comprises a substitution or deletion at a position corresponding to one or more of residues V94, P98, G118, M120, M134, T142, L144, M145, T148, T154, L179, Q204, V206, S210, 1212, T214, L215, G231, T232, V250, Q276, N285, S291, G308, T312, S316, V323, N325, 1333, G334, S343, T349, G360, S380, A381, S386, F411, S413, A416, Q426 and / or A429 in CBH2 from Hypocrea j ecorina (SEQ ID NO: 2). In a first aspect, the invention encompasses a cellulase variant of CBH2, wherein the variant comprises a substitution or deletion in a position corresponding to one or more of the residues V94E, P98L, G118P, M120L, M134G / LV /, T142V, L144G / R / S, M145L, T148Y, T154A, L179A, Q204E, V206L, S210L / R, 1212V, T214M / Y, L215I, G231N, T232V, V250I, Q276L, N285Q, S291G, G308A, T312S, S316P, V323L / N / Y, N325D, I333L, G334A, S343P, T349L / V, G360R, S380T, A381T, S386P, F411Y, S413Y, A416G, Q426E and / or A429T in CBH2 of Hypocrea j ecorina (SEQ ID NO: 2). In a second embodiment, the invention is directed to a variant cellulase of CBH2, wherein the variant comprises a substitution or deletion at a position corresponding to one or more residues V94, P98, G118, M120, M134, T142, M145, T148, T154, L179, Q204, V206, 1212, L215, G231, T232, V250, Q276, N285, S291, G308, T312, S316, V323, N325, 1333, G334, S343, T349, G360, S380, A381, S386, F411, S413, A416, Q426 and / or A429 in CBH2 from Hypocrea j ecorina (SEQ ID NO: 2). In a first aspect, the invention encompasses a variant CBH2 cellulase, wherein the variant comprises a substitution or deletion in a position corresponding to one or more of residues V94E, P98L, G118P, M120L, M134V, T142V, M145L, T148Y, T154A, L179A, Q204E, V206L, 1212V, L215I, G231N, T232V, V250I, Q276L, N285Q, S291G, G308A , T312S, S316P, V323N, N325D, I333L, G334A, S343P, T349L, G360R, S380T, A381T, S386P, F411Y, S413Y, A416G, Q426E and / or A429T in CBH2 from Hypocrea j ecorina (SEQ ID NO: 2) . In a third embodiment, the invention encompasses a cellulose variant of CHB2, wherein the variant comprises a substitution or deletion at a position corresponding to one or more of the residues P98, M134, V206, 1212, T312, S316, F411 and / or S413 in CBH2 from Hypocrea j ecorina (SEQ ID NO: 2). In a first aspect, the invention encompasses a variant of the CBH2 cellulase, wherein the variant comprises a substitution or deletion at a position corresponding to one or more of the residues P98L, M134G / L / V, V206L, 1212V, T312S, S316P , F411Y and / or S413Y in CBH2 from Hypocrea j ecorina (SEQ ID NO: 2). In a fourth embodiment, the invention encompasses a variant of the CBH2 cellulase, wherein the variant comprises a substitution or deletion at a position corresponding to one or more residues in a spatial region, and the spatial region is selected from the group consisting of ( 210, 214), (253, 255, 257, 258), (411, 413, 415), (412, 414, 416), (312, 313), 323, (212, 149, 152), (134, 144) and 98 in CBH2 from Hypocrea jecorina (SEQ ID NO: 2). In a first aspect, the invention encompasses a variant of the cellulase CBH2, wherein the variant is selected from the group consisting of S316P / V323L, S316P / V323Y, V206L / S210R / S316P, V206L / S316P, V206L / S2101L / T214M / S316P, V206L / S210R / T214Y / S316P, M134G / L144G / S316P, M134L / L144R / S316P and M134L / L144S / S316P in CBH2 from Hypocrea j ecorina (SEQ ID N0: 2). In a fifth embodiment, the invention encompasses a cellulase variant of CBH2, wherein the variant comprises a substitution or deletion at a position corresponding to one or more of the residues P98, M134, L144, V206, S210, T214, S316, V323 and / or S413 in CBH2 from Hypocrea j ecorina (SEQ ID NO: 2). In a first aspect, the invention encompasses a cellulase variant of CBH2, wherein the variant comprises a substitution or deletion at a position corresponding to one or more of the residues P98L, M134L / V, L144R, V206L, S210L / R, T214Y, S316P, V323Y and / or S413Y in CBH2 from Hypocrea j ecorina (SEQ ID NO: 2). In a second aspect, the invention encompasses a variant cellulase of CBH2, wherein the variant is selected from the group consisting of 98L / 134V / 206L / 210R / 214Y / 316P / 413Y, 98L / 134L / 144R / 316P / 13Y, 98L / 134L / 144R / 206L / 210R / 214Y / 316P / 413Y, 98L / 134V / 316P / 323Y / 413Y, 98L / 134V / 20617210R / 214Y / 316P / 323Y / 413Y, 98L / 134L / 144R / 316P / 323Y / 413Y, 98L / 134L / 144R / 206L / 210R / 214Y / 316P / 323Y / 413Y, 98L / 13417144R / 210R / 214Y / 316P / 323Y / 413Y and 98L / 134L / 144R / 210L / 214Y / 316P / 323Y / 413Y in CBH2 from Hypocrea j ecorina (SEQ ID NO: 2). In a sixth embodiment, the invention encompasses an isolated nucleic acid encoding a polypeptide having cellobiohydrolase activity. In a first aspect, the invention encompasses an isolated nucleic acid encoding a polypeptide having cellobiohydrolase activity, which polypeptide is a variant of a glycosyl hydrolase of the family 6, and wherein the nucleic acid codes for a substitution in a residue that is sensitive to temperature stress in the polypeptide encoded by the nucleic acid, wherein the variant cellobiohydrolase is derived from the cellobiohydrolase of H. j ecorina. In a second aspect, the invention encompasses an isolated nucleic acid encoding a polypeptide having cellobiohydrolase activity, which polypeptide is a variant of a glycosyl hydrolase of the family 6, and wherein the nucleic acid codes for a substitution in a residue having effects of enzymatic processing capacity on the polypeptide encoded by the nucleic acid, wherein the variant cellobiohydrolase is derived from the cellobiohydrolase of H. jecorina. In a third aspect, The invention encompasses an isolated nucleic acid encoding a polypeptide having cellobiohydrolase activity, which polypeptide is a variant of a glycosylhydrolase of the family 6, and wherein the nucleic acid codes for a substitution in a residue that effects the inhibition of the product. in the polypeptide encoded by nucleic acid, wherein the variant cellobiohydrolase is derived from the cellobiohydrolase of H. j ecorina. In yet another aspect, the invention is directed to an isolated nucleic acid encoding a variant CBH2 cellulase, wherein the variant comprises a substitution at a position corresponding to one or more of the residues V94, P98, G118, M120, M134 , T142, M145, T148, T154, L179, Q204, V206, 1212, L215, G231, T232, V250, Q276, N285, S291, G308, T312, S316, V323, N325, 1333, G334, S343, T349, G360 , S380, A381, S390, F411, S413, A416, Q426 and / or A429 in CBH2 from Hypocrea j ecorina (SEQ ID NO: 2). In a seventh embodiment, the invention is directed to an expression cassette comprising a nucleic acid encoding a variant CBH2. In one aspect, there is a construct comprising the nucleic acid encoding the CBH2 variant operably linked to a regulatory sequence. In an eighth embodiment, the invention is directed to a vector comprising a nucleic acid encoding a CHB2 variant. In one aspect, there is a construct comprising the nucleic acid encoding the CBH2 variant operably linked to a regulatory sequence. In a ninth embodiment, the invention is directed to a host cell transformed with the vector comprising a nucleic acid encoding a variant of CHB2. In a tenth embodiment, the invention is directed to a method for producing a CBH2 variant comprising the steps of: (a) culturing a host cell transformed with the vector comprising a nucleic acid encoding a variant of CBH2, in a suitable culture medium under suitable conditions to produce the CBH2 variant; (b) obtain the CHB2 variant produced. In a tenth embodiment, the invention is directed to a detergent composition comprising a surfactant and a variant of CBH2. In one aspect of this embodiment, the detergent is a laundry detergent. In a second aspect of this embodiment, the detergent is a dishwashing detergent. In a third aspect of this invention, the variant cellulase of CBH2 is used in the treatment of a cellulose-containing textile material, in particular, in the washing with stones or indigo-dyed denim.

In a twelfth embodiment, the invention is directed to a food additive comprising a variant of CBH2. In a thirteenth embodiment, the invention is directed to a method for treating wood pulp, which comprises contacting the wood pulp with a variant of CBH2. In a fourteenth embodiment, the invention is directed to a method for converting the biomass to sugars, which comprises contacting the biomass with a variant of CBH2. In one embodiment, the cellulase is derived from a fungus, bacteria or Actinomycete. In one aspect, the cellulase is derived from a fungus. In another aspect, the fungus is a filamentous fungus. It is preferred that the filamentous fungus belong to Euascomycetes, in particular, Aspergillus spp. , Gliocladi? M spp. , Fusari um spp. , Acremoni um spp. , Mycelioph tora spp. , Verticilli um spp. , Myrothecium spp. , or Penicilli um spp. In a further aspect of this embodiment, the cellulase is a cellobiohydrolase. Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating the preferred modalities of the invention, are given by way of illustration only, since various changes and modifications within the scope and spirit of the invention will become apparent to a person skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE FIGURES Figure 1 is the amino acid sequence (SEQ ID NO: 2) of the wild type Cel6A (CBH2) of H. j ecorina. Figure 2 is the nucleic acid sequence (SEQ ID NO: 1) of wildtype H. jecorina CBH2. Figures 3A-3C show the removal of amino acids from members of the Cel6 family for which there were available crystal structures. The sequences are: -CBH2 from Humi cola insolens, CBH2 from Acremoni um, CBH2 from Agaric? S, CBH2 from Fusarium oxysporum, CBH2 from Hypocrea koningii, CBH2 from Phanerochaete chrysosporum, CBH2 from Talaromyces emersonii, CBH2 from T. reesei (by example, CBH2) of Hypocrea j ecorina, and the consensus sequence. The alignment has been done by Clustal W with an empty space penalty of 10 using the Vector NTI Suite software program. Figure 4 is the pRAXl vector. This vector is based on the plasmid pGAPT2 except for a HindIII fragment of 5259bp of the AMA1 sequence of the genomic DNA fragment of Aspergillus nidulans (Aleksenko and Clutterbuck, Molecular Microbiology 1996 19: 565-574) that was inserted. Base 1 to 1134 contains the promoter of the glucoamylase gene of Aspergill us niger. The bases 3098 to 3356 and 4950 to 4971 contain the glucoamylase terminator of Aspergill us niger. The pyrG gene from Aspergillus nidulans was inserted from 3357 to 4949 as a marker for fungal transformation. There was a multiple cloning site (MCS) within which the genes can be inserted. Figure 5 is the main chain of the pRAXdes2 vector. This vector is based on the plasmid vector pRAXl. An input cassette has been inserted into the pRAXl vector (indicated by a arrow inside the circular plasmid). This cassette contains the recombination sequence attRl and attR2 the selection marker catH and ccdB. The vector has been prepared according to the manual given in Gateway ™ Cloning Technology: version 1 pages 34-38 and can only be replicated in E. coli DB3.1 of Invitrogen; in other E. coli hosts the ccdB gene is lethal. First, a PCR fragment is made with primers that contain the recombination sequences of attBl / 2. This fragment is recombined with pDONR201 (commercially available from Invitrogen); this vector contains the attPl / 2 recombination sequences with catH and ccdB between the recombination sites. The cloning enzymes of BP of Invitrogen are used to recombine the PCR fragment In this so-called INPUT vector, clones with the inserted PCR fragment can be selected at 50 μg / ml kanamycin because clones expressing ccdB do not survive. Now, att sequences are altered and called attLl and attL2. The second step is to recombine this clone with the vector pRAXdes2 (which contains catH and ccdB of attRl and attR2 between the recombination sites). Invitrogen LR clone enzymes are used to recombine the insert from the ENTRADA vector into the target vector. Only pRAXCBH2 vectors are selected using 100 μg / ml ampicillin because ccdB is lethal and the ENTRADA vector is sensitive to ampicillin. By this method, the expression vector is now prepared and can be used to transform A. Niger. Figure 6 provides an illustration of the pRAXdes2cbh2 vector which was used for the expression of the nucleic acids encoding the CBH2 variants in Aspergillus. A nucleic acid encoding a homologous or variant CBH2 enzyme was cloned into the vector by homologous recombination of the att sequences. Figure 7 provides an illustration of the pENTRY-CBH2 vector. Figure 8 is a graph of sugar release, dose dependent, from cellulose swollen with phosphoric acid, by different variants. The variants show a wide range of activity on this substrate. Figure 9 is a bar chart of the ratio of the total (average) sugar produced by one variant, and the total (average) sugar produced by wild-type CBH2 as measured on PASC. Figure 10 is a graph showing the amount of sugar released by varying amounts of the enzyme. The wild type enzyme is denoted as FCA500.3 (open diamonds) and the variant is FCA543 (P98L / M134V / T154A / I212V / S316P / S413Y) (open boxes). The broth from a suppressed strain in CBH2 served as a control. Figure 11 is a bar chart of the proportion of the total (average) sugar produced by the variant and the total (average) sugar produced by the wild type CBH2, as measured on pretreated corn forage. The minimum value of the y-axis scale is 0.6, which represents the value of the total (average) sugar produced by the suppressed strain in CBH2, divided by the total (average) sugar produced by the wild-type CBH2, in combination with the strain deleted in CBH2. A value of 1 represents a level of activity similar to the wild type.

Figure 12 is a graph of a time course experiment. Total sugar released from PASC by a molecule of CBH2 over time at 53 ° C, is shown. The variant is shown as filled triangles (A); of wild type as open pictures (D). Figure 13 is a graph of a time course experiment. The total sugar released from PASC by a molecule of CBH2 over time at 65 ° C, is shown. The variant is shown as filled triangles (• * •); of wild type as open pictures (D). Figure 14 is a graph of a time course experiment. The total sugar released from PASC by a molecule of CBH2 over time at 75 ° C, is shown. The variant is shown as filled triangles (A); of wild type as open pictures (D). Figure 15 is a graph describing the specific operation of a cellulase mixture at 38 ° C. The mixture contains the catalytic core Acidothermus cell ulolyticus and either the wild-type cellobiohydrolases or variants. The wild type is designated 301, 500 which indicates that the wild type CBH1 (for example, 301) and the wild type CBH2 (for example, 500) were used. The variant is designated as 469, 543 which indicates that the variant CBH1 (for example, 469) and the variant CHB2 (for example, 543) were used.

Figure 16 is a graph describing the specific operation of a cellulase mixture at 65 ° C. The mixture contains the catalytic core of Acidothermus cell ulolyti cus and the wild-type or variant cellobiohydrolases. The wild type is designated 301, 500 which indicates that the wild-type CBH1 (for example, 301) and the wild-type CBH2 (for example, 500) were used. The variant is designated as 469, 543 which indicates that the variant CBH1 (for example, 469) and the variant CBH2 (for example, 543) were used. Figure 17 is a graph of the results of the small-scale saccharification conversion assay, at varying temperatures of the variant cellulase mixture, described above in Figure 16. DETAILED DESCRIPTION OF THE INVENTION The invention will now be described in detail by reference only, using the following definitions and examples. All patents and publications, including all sequences described within such patents and publications, herein designated are expressly incorporated by reference. Unless defined otherwise in the present, all technical and scientific terms used herein have the same meaning as that which is commonly understood by a person of ordinary experience in the art. technique to which this invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provides a person skilled in the general dictionary with many of the terms used in this invention. Although any of the methods and materials similar or equivalent to those described herein can be used, in the practice or testing of the invention, preferred methods and materials are described. The numerical ranges are inclusive of the numbers that define the interval. Unless indicated otherwise, nucleic acids are written from left to right in the orientation 5 'to 3'; the amino acid sequences are written from left to right in the amino to carboxyl orientation, respectively. Practitioners are particularly directed to Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL (Second Edition), Cold Spring Harbor Press, Plainview, N.Y., 1989, and Ausubel FM et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1993, for definitions and terms of technique. It should be understood that this invention is not limited to the methodology, protocols and reagents particularly described, since these may vary. The headings provided in this do not they are limitations of the various aspects or embodiments of the invention, which may be taken by reference to the specification as a whole. Consequently, the terms defined immediately below are more fully defined by reference to the specification as a whole. All publications cited herein are expressly incorporated herein by reference for purposes of describing and detailing the compositions and methodologies that may be used in connection with the invention. I. DEFINITIONS The term "polypeptide" as used herein, refers to a compound constituted in a single chain of amino acid residues linked by peptide bonds. The term "protein" as used herein may be synonymous with the term "polypeptide". "Variant" means a protein that is derived from a precursor protein (e.g., the native protein) by adding one or more amino acids to one or both of the C and N termini, replacing one or more amino acids in one or a number of different sites in the amino acid sequence, or the deletion or deletion of one or more amino acids at one or both ends of the protein or at one or more sites in the amino acid sequence. The preparation of an enzyme variant is preferably achieved by modifying a DNA sequence encoding the native protein, transforming the modified DNA sequence into a suitable host, and expressing the modified DNA sequence to form the variant enzyme . The variant CBH2 enzyme of the invention includes peptides comprising altered amino acid sequences compared to an amino acid sequence of precursor enzyme, wherein the variant CBH2 enzyme retains the characteristic cellulolytic nature of the precursor enzyme, but which may have altered properties in some specific aspect For example, a variant CBH2 enzyme can have an increased pH optimum or increased temperature or oxidative stability, but will retain its characteristic cellulolytic activity. It is contemplated that variants according to the present invention may be derived from a DNA fragment encoding a CBH2 cellulase variant enzyme wherein the functional activity of the expressed cellulase variant is conserved. For example, a DNA fragment encoding a cellulase may further include a DNA sequence or portion thereof encoding a hinge or linker linked to the cellulase DNA sequence at either 5 'end. or 3 ', wherein the functional activity of the coded cellulase domain is conserved. The terms variant and derivative may be used interchangeably herein. "Equivalent residues" can also be defined by determination of homology at the level of the tertiary structure for a precursor cellulase, whose tertiary structure has been determined by X-ray crystallography. Equivalent residues are defined as those for which the two coordinates atomic atoms of two or more of the atoms of the main chain of a particular amino acid residue of a cellulase and CBH2 of Hypocrea j ecorina (N on N, CA on CA, C on C and O on O) are within 0.13 nm and preferably 0.1 nm after alignment. The alignment is achieved after the best model has been oriented and placed to give the maximum overlap of atomic coordinates of protein atoms other than hydrogen, from the cellulase in question to the CBH2 of H. j ecorina. The best model is the crystallographic model that gives the lowest R factor for the experimental diffraction data at the highest available resolution.

Equivalent residues that are functionally analogous to a specific CHB2 residue of H. j ecorina are defined as those amino acids of a cellulase that can adopt a conformation such that they alter, modify or contribute to the structure of the protein, substrate binding or catalysis, in a defined manner and attributed to a specific residue of the CBH2 of H. j ecorina. Furthermore, these are those residues of the cellulase (for which a tertiary structure has been obtained by X-ray crystallography) occupying a position analogous to the degree to which, although the atoms of the main chain of the given residue may not meet the criteria of equivalence based on occupying a homologous position, the atomic coordinates of at least two of the atoms of the side chain of the residue are found with 0.13 nm of the atoms of the corresponding side chain of the H. H. ecorina CBH2. The crystal structure of CHB2 of H. j ecorina is shown in Zou et al. (1999) (Ref. 5, supra). The term "nucleic acid molecule" includes the RNA, DNA and cDNA molecules. It will be understood that, as a result of the degeneracy of the genetic code, a plurality of nucleotide sequences encoding a given protein such as CBH2 and / or variants thereof can be produced. The present invention contemplates each possible variant nucleotide sequence, which codes for the variant CBH2, all of which are possible given the degeneracy of the genetic code. A "heterologous" nucleic acid construct or sequence has a portion of the sequence that is not native to the cell for which it is expressed. Heterologous or heterologous, with respect to a control sequence, refers to a control sequence (eg, promoter or enhancer) that does not function naturally to regulate the same gene, the expression of which is currently regulating. In general, heterologous nucleic acid sequences are not endogenous to the cell or part of the genome in which they are present, and have been added to the cell, by infection, transfection, transformation, microinjection, electroporation or the like. A "heterologous" nucleic acid construct can contain a combination of the control sequence / DNA coding sequence, which is the same as or different from a combination of the control sequence / coding sequence of the DNA found in the native cell . As used herein, the term "vector" refers to a nucleic acid construct designed for transfer between different host cells. An "expression vector" refers to a vector that has the ability to incorporate and express a heterologous DNA fragment in a foreign cell. Many vectors of Prokaryotic and eukaryotic expression are commercially available. The selection of appropriate expression vectors is well within the knowledge of those skilled in the art. Accordingly, an "expression cassette" or "expression vector" is a recombinantly or synthetically generated nucleic acid construct, with a series of specified nucleic acid elements, which allow the transcription of a particular nucleic acid in a target cell or objective. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastic DNA, virus or nucleic acid fragment. Typically, the portion of the recombinant expression cassette of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed, and a promoter. As used herein, the term "plasmid" refers to a double-stranded circular DNA construct (ds) used as a cloning vector, and which forms a genetic element of chromosomal self-replication in many bacteria and some eukaryotes. As used herein, the term "nucleotide sequence encoding the selectable marker" refers to a nucleotide sequence that is capable of performing expression in cells and where the Expression of the selectable marker confers to the cells containing the expressed gene, the ability to develop in the presence of a corresponding selective agent, or under corresponding, selective growth conditions. As used herein, the term "promoter" refers to a nucleic acid sequence that functions to direct the transcription of a 3 'gene. The promoter will generally be appropriate for the host cell in which the target gene is being expressed. The promoter together with other transcriptional and translational regulatory nucleic acid sequences (also referred to as "control sequences") are necessary to express a given gene. In general, the transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosomal binding sites, transcription start and stop sequences, translation start and stop sequences and enhancer or activator sequences. "Chimeric gene" or "heterologous nucleic acid construct" as defined herein, refers to a non-native gene (eg, one that has been introduced into a host) that may be composed of parts of different genes, including regulatory elements. The construction of the chimeric gene for the transformation of aThe host cell is typically composed of a transcriptional regulatory region (promoter) operably linked to a sequence encoding the heterologous protein, or in a selectable marker chimeric gene, to a selectable marker gene that encodes a protein that confers, for example, resistance to antibiotics to the transformed cells. A typical chimeric gene of the present invention for transformation within a host cell includes a transcriptional regulatory region that is constitutive or inducible, a sequence encoding the protein, and a terminator sequence. A chimeric gene construct can also include a second DNA sequence encoding a signal peptide if the secretion of the target protein is desired. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, the nucleic acid encoding a secretory guide is operably linked to the DNA for a polypeptide and this is expressed as a pre-protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a sequence of coding if it is placed to facilitate translation. In general, "operably linked" means that the DNA sequences that are linked are contiguous, and in the case of a secretory guide, contiguous and in reading structure. However, the augmentators do not have to be contiguous. The bond is achieved by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adapters, the linkers or primers for PCR are used according to conventional practice. As used herein, the term "gene" means the segment of DNA involved in the production of a polypeptide chain, which may or may not include regions that precede and follow the coding region, eg, the 5 'sequences. untranslated (5 'UTR) or "guide" and the 3' UTR or "back" sequences, as well as the intervention sequences (introns) between the individual coding segments (exons). In general, the nucleic acid molecules encoding variant CBH2 will hybridize, under moderate to highly stringent conditions, to the wild-type sequence provided herein as SEQ ID NO: 1. However, in some cases a nucleotide sequence encoding for CBH2 is used, which possesses a substantially different codon usage, while the protein encoded by the nucleotide sequence encoding GBH2 has the same or substantially the same amino acid sequence as the native protein. For example, the coding sequence can be modified to facilitate the faster expression of GBH2 in a particular prokaryotic or eukaryotic expression system, according to the frequency with which a particular codon is used by the host. Te 'o et al. (FEMS Microbiology Letters 190: 13-19, 2000), for example, describes the optimization of genes for expression in filamentous fungi. A nucleic acid sequence is considered to be "selectively hybridizable" to a reference nucleic acid sequence if the two sequences specifically hybridize with each other under conditions of moderate hybridization and washing up to high demand. Hybridization conditions are based on the melting temperature (Tm) of the complex or nucleic acid binding probe. For example, "peak demand" typically occurs at approximately Tm-5 ° C (5 ° C below the Tm of the probe); "high demand" at approximately 5-10 ° C below the Tm; "moderate" or "intermediate demand" approximately 10-20 ° C below the Tm of the probe, and "low demand" approximately at 20-25 ° C below the Tm. Functionally, the maximum demand conditions may be used to identify the sequences that they have strict identity or almost strict identity with the hybridization probe; while the conditions of high demand are used to identify the sequences that have approximately 80% or more of sequential identity with the probe. Hybridization conditions of moderate and high requirement are well known in the art (see, for example, Sa brook, et al, 1989, Chapters 9 and 11, and in Ausubel, FM, et al., 1993, expressly incorporated by reference at the moment) . An example of high stringency conditions includes hybridization at approximately 42 ° C of 50% formamide, 5X SSC, Denhardt 5X solution, 0.5% SDS and 100 μg / ml denatured carrier DNA, followed by washing twice in 2X SSC and 0.5% SDS at room temperature, and an additional two times at 0. IX SSC and 0.5% SDS at 42 ° C. As used herein, "recombinant" includes reference to a cell or vector, which has been modified by the introduction of a heterologous nucleic acid sequence or that the cell is derived from such a modified cell. Thus, for example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, underexpressed or not fully expressed, as a result of deliberate human intervention.

As used herein, the terms "transformed (a)", "stably transformed (a)", or "transgenic (a)" with reference to a cellular medium, the cell has a non-native nucleic acid sequence (heterologous). ) integrated into its genome or as an episomal plasmid that is maintained across multiple generations. As used herein, the term "expression" refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. The process includes transcription and translation. The term "introduced" in the context of the insertion of a nucleic acid sequence within a cell means "transfection", or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid sequence within of a eukaryotic or prokaryotic cell where the nucleic acid sequence can be incorporated into the genome of the cell (eg, chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomously or transiently expressed replicon (eg, transcribed mRNA). It follows that the term "expression of CBH2" refers to the transcription and translation of the cbh2 gene or variants thereof, the products of which include the precursor RNA, mRNA, the polypeptide, the post-polypeptides. translationally processed, and derivatives thereof, including CBH2 from related species such as Trichoderma koningii, Hypocrea j ecorina (also known as Trichoderma longibra chia tum, Tri choderma reesei or Trichoderma wide) and Hypocrea schweini tzii. By way of example, assays for CBH2 expression include Western blot for CBH2 protein, Northern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR) for mRNA of cbh2, and cellulose initiated with phosphoric acid and assays of PAHBAH as described in the following: (a) PASC: (Karlsson, J. et al. (2001), Eur. J. Biochem, 268, 6498-6507, Wood , T. (1988) in Methods in Enzymology, Vol. 160. Biomass Part a Cellulose and Hemicellulose (Wood, W. &Kellog, S. Eds.), Pp. 19-25, Academic Press, San Diego, CA, USA) and (b) PAHBAH: (Lever, M. (1972) Analytical Biochemistry, 47, 273, Blakeney, A.B. & Mutton, L.L. (1980) Journal of Science of Food and Agriculture, 31, 889, Henry, R.J. (1984) Journal of the Institute of Brewing, 90, 37). The term "alternative splicing" refers to the process by which the multiple isoforms of the polypeptide are generated from a single gene, and involves splicing together the non-consecutive exons during the processing of some, but not all, the transcripts of the gene. In this way, a particular exon can be connected to any of the various alternative exons to form the messenger RNAs. The alternatively spliced mRNAs produce polypeptides ("splice variants") in which some parts are common, while other parts are different. The term "signal sequence" refers to an amino acid sequence in the N-terminal portion of a protein that facilitates the secretion of the mature form of the protein outside the cell. The mature form of the extracellular protein lacks the signal sequence that is cleaved during the secretion process. By the term "host cell" is meant a cell that contains a vector and supports or supports the replication and / or transcription, or transcription and translation (expression) of the expression construct. The host cells for use in the present invention can be prokaryotic cells, such as E. coli, or eukaryotic cells such as yeast cells, plant, insect, amphibian or mammalian cells. In general, the host cells are filamentous fungi. The term "filamentous fungi" refers to any and all filamentous fungi recognized by those of skill in the art. A preferred fungus is selected from the group consisting of Aspergillus, Tri choderma, Fusari um, Chrysospori? M, Penicilli um, Humicola, Neurospora, or alternative sexual forms thereof such as Emerí cella, Hypocrea. It has now been shown that the asexual industrial fungus Trichoderma reesei is a clonal derivative of the Hypocrea jacobina ascomycete. See Kuhls et al., PNAS (1996) 93: 7755-7760. The term "cello-oligosaccharide" refers to groups of oligosaccharides containing from 2 to 8 glucose units and having β-1,4 glucose linkages, for example, cellobiose. The term "cellulase" refers to a category of enzymes capable of hydrolyzing cellulose polymers to shorter cello-oligosaccharide oligomers, cellobiose and / or glucose. Numerous examples of cellulases, such as exoglucanases, exocelobiohydrolases, endoglucanases, and glucosidases have been obtained from cellulolytic organisms, including particularly fungi, plants, and bacteria. CHB2 of Hypocrea j ecorina is a member of family 6 of glucosyl hydrolase (hereinafter Celd) and, specifically, was the first member of that family identified in Hypocrea j ecorina (hereinafter CelßA). Family 6 of glucosyl hydrolase contains endoglucanases and cellobiohydrolases / exoglucanases, and CHB2 is the last. In this way, the phrases CHB2, protein type CBH2 and cellobiohydrolases Cel6 can be used here interchangeably. The term "cellulose binding domain" as used herein refers to a portion of the amino acid sequence of a cellulase or a region of the enzyme that is involved in the cellulose binding activity of a cellulose. cellulase or derivative thereof. The cellulose binding domains generally function by non-covalent linkages of the cellulase to cellulose, a cellulose derivative or other polysaccharide equivalent thereof. The cellulose binding domains allow or facilitate the hydrolysis of cellulose fibers by the structurally distinct, catalytic core region, and function typically independent of the catalytic core. Thus, a cellulose binding domain will not possess significant hydrolytic activity attributable to a catalytic core. In other words, a binding domain to cellulose is a structural element of the tertiary structure of the cellulase enzyme protein, which is distinct from the structural element that possesses catalytic activity. The binding domain to cellulose and the cellulose binding module can be used interchangeably here. As used herein, the term "surfactant" refers to any compound generally recognized in the art as having active surface qualities. In this way, for example, surfactants they comprise anionic, cationic and total non-ionic surfactants such as those commonly found in detergents. Anionic surfactants include linear or branched alkylbenzene sulfonates; alkyl or alkenyl ether sulfates having linear or branched alkyl groups or alkenyl groups; alkyl or alkenyl sulfates; olefinsulfonates; and alcansulfonates. Ampholytic surfactants include sulfates of quaternary ammonium salts, and ampholytic surfactants betaine type. Such ampholytic surfactants have positively and negatively charged groups, in the same molecule. The nonionic surfactants may comprise polyoxyalkylene ethers, as well as higher fatty acid alkanolamides or the alkylene oxide adduct thereof, fatty acid glycerin monoesters, and the like. As used herein, the term "cellulose-containing fabric" refers to any sewn or non-woven fabrics, yarns or fibers made of cotton or cellulose not containing cotton or blends of cellulose containing cotton or not containing cotton, including natural cellulosic materials and man-made cellulosic materials (such as jute, linen, ramia, rayon and lyocell). As used herein, the term "cotton-containing fabric" refers to fabrics sewn or not sewn, the threads or fibers made of pure cotton or cotton blends including woven cotton fabrics, cotton knits, cotton denims, cotton yarns, raw cotton and the like. As used herein, the term "stone wash composition" refers to a formulation for use in washing with stones from fabrics containing cellulose. Stone washing compositions are used to modify fabrics containing cellulose prior to sale, for example, during the manufacturing process. In contrast, the detergent compositions are intended for cleaning soiled garments and are not used during manufacturing processes. As used herein, the term "detergent composition" refers to a mixture that is intended for use in a washing medium for laundry of cellulose-containing fabrics, soiled. In the context of the present invention, such compositions may include, in addition to the cellulases and the surfactants, additional hydrolytic enzymes, additives, bleaching agents, bleach activators, bluing agents and fluorescent dyes, caking inhibitors, agents of masking, antioxidant cellulase activators and solubilizers. As used herein, the term "decrease or elimination in the expression of the cbh2 gene" means that the cbh2 gene has been deleted from the genome and therefore can not be expressed by the recombinant host microorganism; or that the cbh2 or transcript gene has been modified such that a CHB2 functional enzyme is not produced by the host microorganism. The term "variant cbh2 gene" or "variant CBH2" means, respectively, that the nucleic acid sequence of the cbh2 gene from H. jecorina has been altered by the deletion, addition, and / or manipulation of the coding sequence. or the amino acid sequence of the expressed protein has been modified consistent with the invention described herein. As used herein, the term "purification" generally refers to subjecting the transgenic nucleic acid or cells containing protein to biochemical purification and / or column chromatography. As used herein, the term "active" or "biologically active" refers to a biological activity associated with a particular protein and is used interchangeably herein. For example, the enzymatic activity associated with a protease is proteolysis and, thus, an active protease has proteolytic activity. It follows that the biological activity of a given protein refers to any biological activity typically attributed to that protein by those skilled in the art. As used herein, the term "enriched" means that CBH2 is found in a concentration that is higher relative to the concentration of CBH2 found in a wild-type, or naturally occurring, fungal cellulase composition. The terms enriched, elevated and increased can be used here interchangeably. A wild-type fungal cellulase composition is one produced by a fungal source of natural origin and comprising one or more components of BGL, CBH and EG wherein each of these components is found at the ratio produced by the fungal source. Thus, an enriched CBH composition could have CBH at an altered ratio, wherein the ratio of CBH to other cellulase components (eg, EGs, beta-glucosidases and other endoglucanases) is high. This ratio can be increased either by increasing CBH or decreasing (or eliminating) at least one other component by any means known in the art. The term "isolated" or "purified" as used herein, refers to a nucleic acid or amino acid that is removed from at least one component with which it is naturally associated.

Thus, to illustrate, a cellulase system of natural origin can be purified into substantially pure components by well recognized separation techniques, published in the literature, including ion exchange chromatography at a suitable pH, affinity chromatography, chromatography size exclusion and the like. For example, in the exchange chromatography (usually ion exchange chromatography), it is possible to separate the cellulase components by elution with a pH gradient, or a salt gradient, or a pH and a salt gradient. The purified CBH can then be added to the enzyme solution resulting in an enriched CBH solution. It is also possible to raise the amount of CBH produced by a microbe using molecular genetic methods to overexpress the gene encoding CBH, possibly in conjunction with the deletion of one or more genes coding for other cellulases. Fungal cellulases may contain more than one component of CBH. The different components generally have different isoelectric points that allow their separation via ion exchange chromatography and the like. A simple CBH component or a combination of CBH components can be employed in an enzymatic solution.

When used in enzymatic solutions, the homologous or variant CBH2 component is generally added in an amount sufficient to allow the highest release rate of the soluble sugars from the biomass. The amount of the homologous CHB2 component or variant added depends on the type of biomass to be saccharified, which can be easily determined by the person skilled in the art. When employed, the weight percentage of the homologous or variant CHB2 component present in the cellulase composition is preferably between 1 and 100 with the illustrative examples being about 1, preferably about 5, preferably about 10, preferably about 15, or preferably about 20 weight percent to preferably about 25, preferably about 30, preferably about 35, preferably about 40, preferably about 45 or preferably about 50 weight percent. In addition, preferred ranges may be from about 0.5 to about 15 weight percent, 0.5 to about 20 weight percent, from about 1 to about 10 weight percent, from about 1 to about 15 weight percent, of about 1 to about 20 weight percent, from about 1 to about 25 weight percent, of about 5 to about 20 weight percent, about 5 to about 25 weight percent, about 5 to about 30 weight percent, about 5 to about 35 weight percent, about 5 to about 40 percent. weight percent, from about 5 to about 45 percent by weight, from about 5 to about 50 percent by weight, from about 10 to about 20 percent by weight, from about 10 to about 25 percent by weight, of about 10 to about 30 weight percent, from about 10 to about 35 weight percent, from about 10 to about 40 weight percent, from about 10 to about 45 weight percent, from about 10 to about 50 percent by weight, from about 15 to about 60 weight percent, from about 15 to about 65 weight percent, of about 15 to about 70 weight percent, about 15 to about 75 weight percent, about 15 to about 80 weight percent, about 15 to about 85 weight percent, about 15 to about 95 percent; cent in weight. However, when used, the weight percentage of the homologous or variant CBH2 component relative to any EG type components present in the cellulase composition is preferably about 1, preferably about 5, preferably about 10, preferably about 15, or preferably about 20 percent by weight to preferably about 25, preferably about 30, preferably about 35 , preferably about 40, preferably about 45 or preferably about 50 percent by weight. In addition, preferred ranges can be from about 0.5 to about 15 weight percent, from about 0.5 to about 20 weight percent, from about 1 to about 10 weight percent, from about 1 to about 15 weight percent , from about 1 to about 20 weight percent, from about 1 to about 25 weight percent, from about 5 to about 20 weight percent, from about 5 to about 25 weight percent, from about 5 to about 30 weight percent, from about 5 to about 35 weight percent, from about 5 to about 40 weight percent, from about 5 to about 45 weight percent, from about 5 to about 50 weight percent, from about 10 to about 20 weight percent, from about 10 to about 25 weight percent, from about 10 to about 30 percent by weight, from about 10 to about 35 percent by weight, from about 10 to about 40 percent by weight, from about 10 to about 45 percent by weight, from about 10 to about 50 percent by weight, from about 15 to about 20 percent by weight, from about 15 to about 25 percent by weight, from about 15 to about 30 percent by weight, from about 15 to about 35 percent by weight, of about 15 to about 30 weight percent, about 15 to about 45 weight percent, about 15 to about 50 weight percent. II. HOSPEDERAL ORGANISMS Filamentous fungi include all filamentous forms of the subdivision Eumycota and Oomycota. The filamentous fungi are characterized by vegetative mycelium that has a cell wall composed of chitin, glucan, chitosan, mañano and other complex polysaccharides, with vegetative growth by elongation of the hyphae and carbon catabolism that is aerobic bound. In the present invention, the progenitor cell of the filamentous fungus can be a cell of a species of, but not limited to, Tri choderma, eg, Trichoderma longibrachiatum, Trichoderma viride, Trichoderma koningii, Trichoderma harzianum; Penicillium sp.; Humicola sp. , including Humicola insolens and Humicola grísea; Chrysosporium sp. , including C. lucknowense; Gliocladium sp .; Aspergillus sp .; Fusarium sp., Neurospora sp., Hypocrea sp. And Emericella sp. As used herein, the term "Trichoderma" or "Trichoderma sp." It refers to any strains of fungi that have previously been classified as Trichoderma or are currently classified as Trichoderma. In a preferred embodiment, the preferred cell of the filamentous fungus is an Aspergillus niger, Aspergillus awamori, Aspergillus aculeatus, or Aspergillus nidulans. In another preferred embodiment, the progenitor cell of the filamentous fungus is a Trichoderma reesei cell. III. CELLULASES Cellulases are known in the art as enzymes that hydrolyze cellulose (beta-1, -glucan or beta-D-glucosidic bonds) resulting in the formation of glucose, cellobiose, cellooligosaccharides and the like. As described above, cellulases have traditionally been divided into three major classes: endoglucanases (EC 3.2.1.4) ("EG"), exoglucanases or cellobiohydrolases (EC 3.2.1.91) ("CBH") and beta-glucosidases (EC 3.2). .1.21) ("BG"). (Knowles, et al., TIBTECH 5, 255-261, 1987; Schulein, 1988). Certain fungi produce complete cellulase systems that include exo-cellobiohydrolases or cellulase type CBH, endoglucanases or cellulases type EG and beta-glucosidases or cellulases type BG (Schulein, 1988). However, sometimes these systems lack the CBH type cellulases and the bacterial cellulases also typically include little or no CBH type cellulases. In addition, it has been shown that the EG components and CBH components interact synergistically to more efficiently degrade cellulose. See, for example, Wood, 1985. The different components, for example the different endoglucanases and exocelobiohydrolases in a system of multiple components or complete cellulase, generally have different properties, such as the isoelectric point, molecular weight, degree of glycosylation, specificity of substrate and patterns of enzymatic action. It is believed that endoglucanase-type cellulases hydrolyse internal beta-1, 4-glycosidic bonds in regions of low crystallinity of cellulose, and exo-cellobiohydrolase-type cellulases hydrolyze cellobiose from the reducing or non-reducing end of cellulose. It follows that the action of the endoglucanase components can greatly facilitate the action of the exo-cellobiohydrolases by creating new extremes of chain that are recognized by the exo-cellobiohydrolase components. In addition, beta-glucosidase type cells have been shown to catalyze the hydrolysis of alkyl and / or aryl beta-D-glucosides such as methyl beta-D-glucosides and p-nitrophenyl glucoside, as well as glycosides containing only carbohydrate residues, such as cellobiose. This produces glucose as the only product for the microorganism and reduces or eliminates the cellobiose that inhibits cellobiohydrolases and endoglucanases. Cellulases also find a number of uses in detergent compositions, including to increase cleaning ability, as a softening agent and to improve the feel of cotton fabrics (Hemmpel, ITB Dyeing / Printing / Finishing 3: 5-14 , 1991, Tyndall, Textile Chemist and Colorist 24: 23-26, 1992, Kumar et al., Textile Chemist and Colorist, 29: 37-42, 1997). While the mechanism is not part of the invention, the cellulase color softening and restoration properties have been attributed to the alkaline endoglucanase components in the cellulase compositions, as exemplified in U.S. Patent Nos. 5,648,263 , 5,691,178, and 5,776,757, which disclose that detergent compositions containing a cellulase composition enriched in an endoglucanase component Alkaline-specific, impart color restoration and improved softening to treated garments, compared to cellulase compositions not enriched in such a component. In addition, the use of such alkaline glucanase components in detergent compositions has been shown to supplement the pH requirements of the detergent composition (for example, by showing maximum activity at an alkaline pH of 7.5 to 10, as described in the US Patent Nos. United States Nos. 5,648,263, 5,691,178, and 5,776,757). Cellulase compositions have also been shown to degrade cotton-containing fabrics, resulting in reduced loss of strength in the fabric (U.S. Patent No. 4,822,516), contributing to the reluctance to use cellulase compositions in commercial detergent applications. . It has been suggested that cellulase compositions comprising endoglucanase components show reduced strength loss for cotton-containing fabrics, compared to compositions comprising a complete cellulase system. It has also been shown that cellulases are useful in the degradation of biomass from cellulase to ethanol (where cellulase degrades cellulose to glucose, and yeasts and other microbes ferment in addition glucose to ethanol), in the mechanical treatment of pasta paper mill (Pere et al., In Proc. Tappi Pulping Conf., Nashville, TN, 27-31, pp. 693-696, 1996), for use as a food additive (WO 91/04673) and in the wet milling of grains. Most CBHs and EGs have a multidomain structure consisting of a core domain separated from a cellulose binding domain (CBD) by a linker peptide (Suurnakki et al., 2000). The core domain contains the active site, whereas CBD interacts with cellulose by binding the enzyme to it (van Tilbeurgh et al., FEBS Lett 204: 223-227, 1986, Tomme et al., Eur. J Biochem 170: 575-581, 1988). CBDs are particularly important in the hydrolysis of crystalline cellulose. It has been shown that the ability of cellobiohydrolases to degrade crystalline cellulose decreases clearly when CBD is absent (Linder and Teeri, J. Biotechnol 57: 15-28, 1997). However, the exact role and mechanism of action of the CBDs is still a matter of speculation. It has been suggested that CBD increases enzymatic activity merely by increasing the concentration of effective enzyme on the surface of cellulose (Stahlberg et al., Bio / Technol 9: 286-290, 1991), and / or by freezing the simple cellulose chains of the cellulose surface (Tormo et al., EMBO J. Vol. 15, No. 21, pp. 5739-5751, 1996). Most studies concerning the effects of cellulase domains on different substrates have been carried out with core proteins of the cellobiohydrolases, since their core proteins can be easily produced by limited proteolysis with papain (Tom et al., 1988). Numerous cellulases have been described in the scientific literature, examples of which include: Trichoderma reesei. Shoemaker, S. et al., Bio / Technology, 1: 691-696, 1983, which describes CBH1; Teeri, T. et al., Gene, 51: 43-52, 1987, which describes CHB2. Cellulase from different species of Tri choderma have also been described, for example, Ooi et al., Nucleic Acids Research, Vol. 18, No. 19, 1990, which describes the cDNA sequence encoding the endoglucanase Fl-CMC produced by Aspergill us aculea tus; Kawaguchi T et al., Gene 173 (2): 287-8, 1996, which describes the cloning and sequencing of the cDNA encoding Aspergill beta-glucosidase 1 us aculea tus; Sakamoto et al., Curr. Genet 27: 435-439, 1995, which describes the cDNA sequence coding for the endoglucanase CMCase-1 from Aspergill us kawa chii IFO 4308; Saarilahti et al., Gene 90: 9-14, 1990, which describes an endoglucanase from Erwinia carotovara; Spilliaert R, et al., Eur J Biochem. 224 (3): 923-30, 1994, which describes cloning sequencing of bglA, which codes for a thermostable beta-glucanase from Rhodothermus marinus; and Halldorsdottir S et al., Appl Microbiol Biotechnol. 49 (3): 277-84, 1998, which describes the cloning, sequencing and overexpression of a Rhodothermus marinus gene that codes for a thermostable cellulase of family 12 of glucosyl hydrolase. However, there remains a need for the identification and characterization of new cellulases, with improved properties, such as improved performance under thermal stress conditions or in the presence of surfactants, increased specific activity, altered pattern of substrate cleavage, and / or high level expression in vi tro. The development of new and improved cellulase compositions comprising varying amounts of the CBH, type EG and type BG cellulases is of interest for use: (1) in detergent compositions that exhibit improved cleaning ability, function as a softening agent and / or improve the feel of cotton fabrics (for example, "stone washing" or "bio-polishing"); (2) in compositions for degrading wood pulp or other biomass in sugars (for example, for the production of bioethanol); and / or (3) in food compositions. IV. MOLECULAR BIOLOGY In one embodiment, this invention provides for the expression of variant cbh2 genes under the control of a functional promoter in a filamentous fungus. Therefore, this invention relies on routine techniques in the field of recombinant genetics. The basic texts that describe the general methods for use in this invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Ausubel et al., eds., Current Protocols in Molecular Biology (1994)). Methods for Identifying Homologous Genes of cbh.2 The nucleic acid sequence for wild type H. j ecorin CBH2 is shown in Figure 1. The invention, in one aspect, encompasses a nucleic acid molecule encoding a homologue of CBH2 described herein. The nucleic acid can be a DNA molecule. Techniques that can be used to isolate the DNA sequences encoding CBH2 are well known in the art, but are not limited to, cDNA and / or genomic library selection with a homologous DNA probe and selection of expression with activity assays or antibodies against CBH2. Any of these methods can be found in Sambrook, et al. or in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, F. Ausubel, et al., ed. Greene Publishing and Wiley-Interscience, New York (1987) ("Ausubel"). Mutation Methods of the cbh2 Nucleic Acid Sequences Any method known in the art that can introduce mutations is contemplated by the present invention.

The present invention relates to the expression, purification and / or isolation and use of CBH2 variant. These enzymes are preferably prepared by recombinant methods using the cbh2 gene of H. jecorina. The fermentation broth can be used with or without purification. After isolation and cloning of the H. jecorin cbh2 gene, other methods known in the art, such as site-directed mutagenesis, are used to perform the substitutions, additions or deletions corresponding to the substituted amino acids in the CHB2 variant. expressed. Again, site-directed mutagenesis and other methods of incorporating amino acid changes in proteins expressed at the DNA level can be found in Sambrook et al. and Ausubel et al. The DNA encoding a variant of the amino acid sequence of H. j ecorina CBH2 is prepared by a variety of methods known in the art. These methods include, but are not limited to, the preparation by site-directed mutagenesis (or mediated by the oligonucleotide), PCR mutagenesis, and cassette mutagenesis of an earlier prepared DNA coding for H. j ecorina CBH2. Site-directed mutagenesis is a preferred method for preparing substitution variants. This technique is well known in the art (see, for example, Cárter et al. Nucleic Acids Res. 13: 4431-4443 (1985) and Kunkel et al., Proc. Nati Acad. Sci. USA 82: 488 (1987)). In summary, in carrying out site-directed mutagenesis of DNA, the initial DNA is first altered by hybridization of an oligonucleotide encoding the desired mutation to a single strand of such an initial DNA. After hybridization, a DNA polymerase is used to synthesize a second whole strand, using the hybridized oligonucleotide as a primer, and using the single strand of the initial DNA as a template. In this way, the oligonucleotide coding for the desired mutation is incorporated into the resulting double-stranded DNA. PCR mutagenesis is also suitable for the preparation of variants of the amino acid sequence of the initial polypeptide, for example CBH2 of H. j ecorina. See Higuchi, in PCR Protocols, p. 177-183 (Academic Press, 1990); and Vallette et al., Nuc. Acids Res. 17: 723-733 (1989). See also for example Cadwell et al., PCR Methods and Applications, Vol. 2, 28-33 (1992). In summary, when small amounts of the template DNA are used as the initial material in a PCR, primers that differ slightly in sequence from the corresponding region in a template DNA, can be used to generate relatively large amounts of a specific DNA fragment that it differs from the template sequence only in the positions where the primers differ from the template. Yet another method for preparing the variants, cassette mutagenesis, is based on the technique described by Wells et al., Gene 34: 315-323 (1985). The starting material is the plasmid (or other vector) comprising the DNA of the initial polypeptide to be mutated. The codon (s) in the initial DNA that is to be mutated are identified. There must be a unique restriction endonuclease site on each side of the identified mutation site (s). If no such restriction sites exist, they can be generated using the oligonucleotide-mediated mutagenesis method, described above, to be introduced at the appropriate sites in the initial polypeptide DNA. The plasmid DNA is cut at these sites to linearize it. A double-stranded oligonucleotide encoding the DNA sequence between the restriction sites, but containing the desired mutation (s), is synthesized using standard procedures, wherein the two strands of the oligonucleotide are synthesized separately and then hybridized to each other using techniques standards This double-stranded oligonucleotide is termed as the cassette. This cassette is designed to have 5 'and 3' ends that are compatible with the ends of the linearized plasmid, such that this can be directly linked to the plasmid. This plasmid now contains the mutated DNA sequence. Alternatively or additionally, the desired amino acid sequence encoding a variant of CBH2 can be determined, and a nucleic acid sequence encoding such a variant amino acid sequence, can be generated synthetically. The CBH2 variants prepared here may be subjected to further modifications, sometimes depending on the intended use of the cellulase. Such modifications may involve further alteration of the amino acid sequence, fusion to one or more heterologous polypeptides and / or covalent modifications. V. Nucleic Acids of cbh.2 and Polypeptides CBH2 A. Nucleic Acids Type cbh.2 Variant The nucleic acid sequence for cbh2 of wild-type H. jecorin is shown in Figure 1. The invention encompasses a nucleic acid molecule which codes for the variant cellulases described herein. The nucleic acid can be a DNA molecule. After isolation and cloning of cbh2, other methods known in the art, such as site-directed mutagenesis, are used to perform substitutions, additions or deletions corresponding to the substituted amino acids in the variant CBH2 expressed.

Again, site-directed mutagenesis and other methods of incorporating amino acid changes in proteins expressed at the DNA level can be found in Sambrook et al. and Ausubel et al. After the DNA sequences encoding the CBH2 variants have been cloned into the DNA constructs, the DNA is used to transform the microorganisms. The microorganism that is to be transformed for the purpose of expressing a variant of CBH2 according to the present invention, can advantageously comprise a strain derived from Tri choderma sp. Thus, a preferred way to prepare the variant cellulases of CBH2 according to the present invention comprises transforming a host cell of Trichoderma sp. with a DNA construct comprising at least one DNA fragment encoding a portion or all of the variant CBH2. The construction of DNA in general will be functionally linked to a promoter. The transformed host cell is then developed under conditions to express the desired protein. Subsequently, the desired protein product can be purified to substantial homogeneity. However, it may in fact happen that the best expression vehicle for a given DNA encoding a variant of CBH2 may differ from H. j ecorina. In this way, it can happen that it will be more advantageous to express a protein in a transformation host that has phylogenetic similarity to the source organism, for the CBH2 variant. In an alternative mode, Aspergill us niger can be used as an expression vehicle. For a description of transformation techniques with A. niger, see WO 98/31821, the description of which is incorporated by reference herein in its entirety. Accordingly, the present description of an expression system of Aspergillus spp. it is provided for illustrative purposes only, and as an option to express the CHB2 variant of the invention. A person skilled in the art, however, may be inclined to express the DNA encoding the CBH2 variant in a different host cell, if appropriate, it should be understood that the source of the variant CBH2 could be considered in the determination of the optimal expression host. In addition, the skilled worker in the field will be able to select the best expression system for a particular gene through routine techniques using the tools available in the field. B. CBH2 Polypeptides Variant The amino acid sequence for wild type H. j ecorina CBH2 is shown in Figure 1. CBH2 variant polypeptides comprise a substitution or deletion at a position corresponding to one or more Residues V94, P98, G118, M120, M134, T142, L144, M145, T148, T154, L179, Q204, V206, S210, 1212, T214, L215, G231, T232, V250, Q276, N285, S291, G308, T312 , S316, V323, N325, 1333, G334, S343, T349, G360, S380, A381, S386, F411, S413, A416, Q426 and / or A429 in CBH2 from Hypocrea j ecorina (SEQ ID N0: 2). In one aspect, the invention relates to an CHB2 enzyme isolated from the Cell family that has at least one substitution or deletion of the amino acid residue in a region selected from the group consisting of (1) from position 92 to 100, (2) ) 115-123, (3) 140-155, (4) 160-180, (5) 198-218, (6) 228-235, (7) 240-260, (8) 275-295, (9) 305-318, (10) 322-335, (11) 340-350, (12) 360-370, (13) 378-390 and (14) 410-430. In still another aspect, the invention relates to a CBH2 enzyme isolated from the family CeldA having at least one substitution of the amino acid residue in a region selected from the group consisting of (1) from position 92 to 100, (2) 115-123, (3) 140-155, (4) 160-180, (5) 198-218, (6) 228-235, (7) 240-260, (8) 275-295, (9) 305 -318, (10) 322-335, (11) 340-350, (12) 360-370, (13) 378-390 and (14) 410-430. The CHB2 variant of this invention has amino acid sequences that are derived from the amino acid sequence of a CBH2 precursor. The amino acid sequence of the CBH2 variant differs from the amino acid sequence of the CBH2 precursor by substitution, deletion or insertion of one or more amino acids of the precursor amino acid sequence. In a preferred embodiment, the precursor CBH2 is CBH2 from Hypocrea j ecorin. The mature amino acid sequence of H. j ecorina CBH2 is shown in Figure 1. Thus, this invention is directed to variants of CBH2 that contain amino acid residues at positions that are equivalent to the particular residue identified in H CBH2. . j ecorina. A residue (amino acid) of a CHB2 homologue is equivalent to a CBH2 residue of Hypocrea j ecorin if it is either homologous (eg, corresponding in position in the primary or secondary structure) or functionally analogous to a specific residue or specific portion of that residue in Hypocrea j ecorine CBH2 (for example, having the same or a similar functional ability to combine, react or interact chemically or structurally). As used herein, the numbering is intended to correspond to that of the mature amino acid sequence of CBH2, as illustrated in Figure 1. In addition to the sites within the precursor CBH2, the specific residues in precursor CHB2 corresponding to the amino acid positions that are responsible for the instability when the precursor CHB2 is under thermal stress, are identified herein for substitution or deletion. The amino acid position number (for example, +51) refers to the assigned number to the mature sequence of CBH2 of Hypocrea j ecorina presented in Figure 1. Alignment of the amino acid sequences to determine homology is preferably determined by the use of a "sequence comparison algorithm". The optimal alignment of the sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Ma th. 2: 482 (1981), but the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970), by means of the search method for similarity of Pearson & Lipman, Proc. Nati Acad. Sci. USA 85: 2444 (1988), through computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI) or by visual inspection. Visual inspection can use graphic packages such as, for example, MOE by Chemical Computing Group, Montreal Canada. An example of an algorithm that is suitable for determining sequential similarity is the BLAST algorithm, which is described in Altschul, et al, J. Mol. Biol. 215: 403-410 (1990). Software to perform BLAST analyzes is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). East The algorithm first involves the identification of highly qualified sequence pairs (HSPs) by identifying short words of length W in the search sequence that either mesh or satisfy some positive value threshold T when they are aligned with a word of the same length in a database sequence. These initial neighbor word hits act as starting points to find longer HSPs that contain them. The word hits are expanded in both directions along each of the two sequences that are compared, so that the cumulative alignment qualification can be increased. The extension of word hits is stopped when: the cumulative alignment score decreased by the amount X from a maximum value achieved; the cumulative rating goes up to zero or below; or the end of any sequence is reached. The parameters W, T and X of the algorithm determine the sensitivity and speed of the alignment. The BLAST program uses as omissions a word length (W) of 11, the qualification matrix BLOSUM62 (see Henikoff &Henikoff, Proc. Nati, Acad. ScL USA 89: 10915 (1989)) alignments (B) of 50 , expectation (E) of 10, M'5, N'4, and a comparison of both strands. The BLAST algorithm then performs a statistical analysis of the similarity between the two sequences (see, for example, Karlin & Altschul, Proc. Nati Acad. ScL USA 90: 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences could probably occur. For example, an amino acid sequence is considered similar to a protease if the smallest sum probability in a comparison of the test amino acid sequence to a protease amino acid sequence is less than 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. For purposes of the present invention, the degree of identity can be adequately determined by means of computer programs known in the art, such as GAP provided in the GCG program package (Wisconsin Package Program Manual, Version 8, August 1994 , Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman, SB and Wunsch, CD, (1970), Journal of Molecular Biology, 48, 443-45), using GAP with the following settings for the comparison of the nucleotide sequence: penalty for creation of empty space (GAP) of 5.0 and penalty for extension of empty space (GAP) of 0.3.

Sequence searches are typically carried out using the BLASTN algorithm when evaluating a given nucleic acid sequence relative to the nucleic acid sequences in the GenBank DNA sequences and other public databases. The BLASTX program is preferred to search for nucleic acid sequences that have been translated in all reading structures against the amino acid sequences in the GenBank Protein Sequences and other public databases. BLASTN and BLASTX are run using default parameters of an open empty space penalty of 11.0 and an extended empty space penalty of 1.0, and use the BLOSUM62 matrix. (See, for example, Altschul, et al, 1997). Additional specific strategies to modify the stability of CBH2 cellulases are provided below: (1) The decrease in the entropy of the unfolding of the main chain can introduce stability to the enzyme. For example, the introduction of proline residues can significantly stabilize the protein by decreasing the entropy of the unfolding (see, for example, Watanabe, et al., Eur. J. Biochem. 226: 277-283. (1994)). Similarly, the glycine residues have no β-carbon, and thus have a substantially greater conformational freedom of the main chain, which many other waste. The replacement of glycine, preferably with alanine, can reduce the entropy of unfolding and improve stability (see, for example, Matthews, et al., Proc. Nati, Acad. Sci. USA 84; 6663-6667 (1987)) . In addition, by shortening the external loops it may be possible to improve the stability. It has been observed that proteins produced by hyperthermophiles have shorter external loops than their mesophilic counterparts (see, for example, Russel, et al., Current Opinions in Biotechnology 6: 370-374 (1995)). The introduction of disulfide bonds can also be effective in stabilizing different tertiary structures relative to each other. Thus, the introduction of the cysteines in the accessible residues to the existing cysteines or the introduction of pairs of cysteines that could form disulfide bonds could alter the stability of a variant of CBH2. (2) The decrease of the internal cavities by the increase of the side chain hydrophobicity can alter the stability of an enzyme. The reduction of the number and volume of internal cavities increases the stability of the enzyme by maximizing hydrophobic interactions and reducing packaging defects (see, for example, Matthews, Ann.Rev. Biochem. 62: 139-160 (1993). ); Burley, et al., Science 229: 23- 29 (1985); Zuber, Biophys. Chem. 29: 171-179 (1988); Kellis, et al., Nature 333: 784-786 (1988)). It is known that multimeric proteins from thermophiles often have more hydrophobic subunit interconnections with greater surface complementarity than their mesophilic counterparts (Russel et al., Supra). It is believed that this principle is applicable to monomeric protein domain interfaces or interconnections. Specific substitutions that can improve stability by increasing hydrophobicity include lysine to arginine, serine to alanine and threonine to alanine (Russel et al., Supra). Modification by substitution to alanine or proline, can increase the size of the side chain with the resulting reduction in the cavities, better packaging and increased hydrophobicity. The substitutions to reduce the size of the cavity, increase the hydrophobicity and improve the complementarity of the interfaces between the CBH2 domains, can improve the stability of the enzyme. Specifically, modification of the specific residue at these positions with a different residue selected from either phenylalanine, tryptophan, tyrosine, leucine and isoleucine, can improve performance. (3) The load balance in the rigid secondary structure, for example, the helices and the ß turns can improve stability. For example, neutralization of partial positive charges on an N-terminus of the negatively charged helix on the aspartic acid can improve the stability of the structure (see, for example, Eriksson, et al., Science 255: 178-183 (1992)). Similarly, the neutralization of partial negative charges on the C-terminus of the positively charged helix can improve stability. The removal of the positive charge from the interaction with the N-terminus of the peptide in the ß-turns should be effective in conferring the stability of the tertiary structure. Substitution with a non-positively charged residue could eliminate an unfavorable positive charge so as not to interact with an amide nitrogen present in the spin. (4) The introduction of salt bridges and hydrogen bonds to stabilize tertiary structures can be effective. For example, interactions of ion pairs, for example, between aspartic acid and glutamic acid and lysine, arginine or histidine, can introduce strong stabilizing effects and can be used to couple different elements of tertiary structure with a resultant improvement in the thermostability. In addition, increases in the number of hydrogen bonds of charged residue / unloaded residue, and the number of hydrogen bonds in general, can improve thermostability (see, for example, Tanner, et al., Biochemistry 35: 2597- 2609 (nineteen ninety six)). Substitution with aspartic acid, asparagine, glutamic acid or glutamine can introduce a hydrogen bond with a main chain amine. Substitution with arginine can improve a salt bridge and introduce a hydrogen bond within a carbonyl of the main chain. (5) Prevent thermolabile residues in general from increasing thermal stability. For example, asparagine and glutamine are susceptible to deamidation, and cysteine is susceptible to oxidation at high temperatures. Reducing the number of these residues in sensitive positions can result in improved thermostability (Russel et al., Supra). Substitution or deletion by any residue other than glutamine or cysteine may increase stability by avoidance of a thermolabile residue. (6) The stabilization or destabilization of the binding of a ligand that confers modified stability to CBH2 variants. For example, a component of the matrix in which the CBH2 variants of this invention are used can be linked to a specific surfactant / thermal sensitivity site of the CBH2 variant. By modifying the site through substitution, the link of the component to the variant can be reinforced or decreased. For example, a non-aromatic residue in the crack or crevice The binding of CBH2 can be substituted with phenylalanine or tyrosine to introduce stabilization of the aromatic side chain, where the interaction of the cellulose substrate can interact favorably with the benzyl rings, increasing the stability of the variant CBH2. (7) Increasing the electronegativity of any surfactant ligands / thermal sensitivity can improve stability under thermal or surfactant stress. For example, substitution with phenylalanine or tyrosine may increase the electronegativity of D (aspartate) residues by improving the protection against the solvent, thereby improving stability. SAW . Expression of Recombinant Variants of CBH2 The methods of the invention rely on the use of cells to express the CBH2 variant, with no particular method of expression of CBH2 required. CHB2 is preferably secreted from the cells. The invention provides host cells that have been transduced, transformed or transfected with an expression vector comprising a variant sequence of the nucleic acid encoding CBH. The culture conditions, such as temperature, pH and the like, are those previously used for the progenitor host cell before transduction, transformation or transfection and will be apparent to those skilled in the art. technique . In one method, a filamentous fungal cell or yeast cell is transfected with an expression vector having a promoter or biologically active promoter fragment or one or more (eg, a series) of enhancers that function in the host cell line, operably linked to a segment of DNA encoding CBH2, such that CBH2 is expressed in the cell line. A. Nucleic Acid Constructs / Expression Vectors Natural or synthetic polynucleotide fragments encoding CBH2 ("nucleic acid sequence encoding CBH2") can be incorporated into heterologous nucleic acid constructs or vectors capable of performing the introduction inside and replication in, a filamentous fungus or yeast cell. The vectors and methods described herein are suitable for use in host cells for the expression of CBH2. Any vector can be used as long as it is replicable and viable in the cells into which it is introduced. Large numbers of suitable vectors and promoters are known to those skilled in the art, and are commercially available. Cloning and expression vectors are also described in Sambrook et al., 1989, Ausubel FM et al., 1989, and Strathern et al., The Molecular Biology of the Yeast Saccharomyces, 1981, each of which is expressly incorporated by reference herein. Expression vectors suitable for fungi are described in van den Hondel, C.A.M.J.J. et al. (1991) in: Bennett, J.W. and Lasure, LL (eds.) More Gene Manipulations in Fungí. Academic Press, p. 396-428. The appropriate sequence of the DNA can be inserted into a plasmid or vector (collectively referred to herein as "vectors") by a variety of methods. In general, the DNA sequence is inserted into one or several appropriate restriction endonuclease sites, by standard procedures. Such procedures and related subcloning procedures are considered within the scope of the knowledge of those skilled in the art. Recombinant filamentous fungi comprising the coding sequence for the CBH2 variant, can be produced by introducing a heterologous nucleic acid construct comprising the sequence encoding the variant of CHB2, into the cells of a selected strain of the filamentous fungi. Once the desired form of a variant cbh2 nucleic acid sequence is obtained, it can be modified in a variety of ways. Where the sequence involves flanking regions of non-coding, the flanking regions can be subjected to resection, mutagenesis, etc. In this way, transitions, transversions, deletions and insertions can be made on the sequence of natural origin. A sequence encoding selected cbh2 variant can be inserted into a suitable vector according to well-known recombinant techniques, and used to transform filamentous fungi capable of performing CBH2 expression. Due to the inherent degeneracy of the genetic code, other nucleic acid sequences that encode substantially for the same sequence or a functionally equivalent functional amino acid sequence can be used to clone and express the variant CBH2. Therefore, it is appreciated that such substitutions in the coding region fall within the sequence variants covered by the present invention. Any and all of these sequence variants can be used in the same manner as described herein for a progenitor nucleic acid sequence encoding CBH2. The present invention also includes recombinant nucleic acid constructs comprising one or more of the nucleic acid sequences encoding the CBH2 variant, as described above. The constructs comprise a vector, such as a plasmid or viral vector, within which a sequence of the invention has been inserted, in a forward or inverse orientation. Heterologous nucleic acid constructs may include the coding sequence for the cbh2 variant: (i) in isolation; (ii) in combination with the additional coding sequences; such as the sequences encoding the fusion protein or for the signal peptide, wherein the sequence encoding cbh2 is the dominant coding sequence; (iii) in combination with the non-coding sequences, such as the introns and the control elements, such as the promoter and terminator elements or the 5 'and / or 3' untranslated regions, effective for the expression of the sequence of coding in a suitable host; and / or (iv) in a vector or host environment in which the sequence encoding cbh2 is a heterologous gene. In one aspect of the present invention, a heterologous nucleic acid construct is employed to transfer a variant CBH2 coding nucleic acid sequence, within an in vitro cell, with filamentous fungal lines and established, preferred yeasts. For the long-term production of the variant CBH2, stable expression is preferred. It follows that any effective method to generate stable transformants, can be used in the practice of the invention. Appropriate vectors are typically equipped with a nucleic acid sequence encoding the selectable marker, insertion sites, and suitable control elements, such as the promoter and terminator sequences. The vector may comprise regulatory sequences, including, for example, non-coding sequences, such as introns and control elements, for example promoter and terminator elements or 5 'and / or 3' untranslated regions, effective for the expression of the coding sequence in the host cells (and / or in a vector or host cell environment in which the sequence encoding the soluble protein antigen, modified, is not normally expressed), operably linked to the sequence of coding. Large numbers of suitable vectors and promoters are known to those skilled in the art, many of which are commercially available and / or are described in Sambrook et al., (Supra). Exemplary promoters include constitutive promoters and inductive promoters, examples of which include a CMV promoter, an SV40 early promoter, an RSV promoter, an EF-la promoter, a promoter that contains the tet-responsive element (TRE) in the system from tet ignition or tet off as described (Clone Tech and BASF), the promoter of beta-actin and the promoter of metallothionein that can be supraregulated by the addition of certain metal salts. A promoter sequence is a DNA sequence that is recognized by the particular filamentous fungus for expression purposes. This was operably linked to the DNA sequence encoding a variant CBH2 polypeptide. Such a link comprises the positioning of the promoter with respect to the start codon of the DNA sequence encoding the variant CBH2 polypeptide in the described expression vectors. The promoter sequence contains the transcriptional and translational control sequence which are mediators of expression of the variant CBH2 polypeptide. Examples include promoters from genes encoding glucoamylase, alpha-amylase, or alpha-glucosidase from Aspergillus niger, A awamori or A. oryzae; the gpdA or trpC genes of A. nidulans; the genes of cbhl or trpl of Neurospora crassa; the genes that code for the aspartic proteinase of A. niger or Rhizomucor miehei; the genes that code for cbhl, cbh2, egl l, egl2 or other cellulases of H. j ecorina (T. reesei). The choice of the suitable selectable marker will depend on the host cell, and the appropriate markers for different hosts are well known in the art. Typical selectable marker genes include argB of A. nidulans or T. reesei, amdS from A. nidulans, pyr4 from Neurospora crassa or T. reesei, pyrG from Aspergill us niger or A. nidulans. Additional exemplary selectable markers include, but are not limited to, trpc, trpl, OÜC31, niaD or leu2, which are included in the heterologous nucleic acid constructs used to transform a mutant strain such as trp-, pyr-, leu- and similar. Such selectable markers confer transformants the ability to transform a metabolite that is usually not metabolized by filamentous fungi. For example, the amdS gene of H. j ecorina that codes for the enzyme acetamidase that allows transforming cells to develop on acetamide as a source of nitrogen. The selectable marker (eg, pyrG) can restore the ability of an autotrophic mutant strain to develop on a selective minimal medium or the selectable marker (eg, olic31) can confer transformants the ability to develop in the presence of an inhibitory drug. or antibiotic. The sequence encoding the selectable marker is cloned into any suitable plasmid using methods generally employed in the art. Exemplary plasmids include pUC18, pBR322, pRAX and pUClOO. Plasmid pRAX contains the AMA1 sequences of A. nidulans, which makes it possible to replicate in A. niger. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are fully explained in the literature. See, for example, Sambrook et al., 1989; Freshney, Animal Cell Culture, 1987; Ausubel, et al., 1993; and Coligan et al., Current Protocols in Immunology, 1991. B. Host Cells and Culture Conditions for the Production of CBH2 (i) Filamentous Fungi In this way, the present invention provides filamentous fungi comprising cells that have been modified, selected and cultured in an effective manner to result in the production of the CBH2 variant or the expression thereof in relation to the corresponding non-transformed progenitor fungi. Examples of progenitor filamentous fungal species that can be treated and / or modified for expression of variant CBH2 include, but are not limited to Trichoderma, eg, Tri choderma reesei, Tri choderma longibra chia tum, Tri choderma viride, Trichoderma koningii; Penicilli um sp. , Humi cola sp. , including H? mycola insolens; Aspergill us sp. , Chrysospori um sp. , Fusarium sp. , Hypocrea sp. , and Emericella sp. Cells expressing CHB2 are cultured under conditions typically employed to culture the progenitor fungal line. In general, the cells are cultured in a standard medium containing physiological salts and nutrients, such as described in Pourquie, J. et al., Biochemistry and Genetics of Cellulose Degradation, eds. Aubert, J. P. et al., Academic Press, p. 71-86, 1988 and Limen, M. et al., Appl. Environ. Microbiol. 63: 1298-1306, 1997. The culture conditions are also standard, for example, the cultures are incubated at 28 ° C in shaking cultures or agitators with stirring until the desired levels of CBH2 expression are reached. The preferred culture conditions for a given filamentous fungus can be found in the scientific literature and / or from the source of the fungi such as the American Type Culture Collection (ATCC; www.atcc.org/) After it has been established. growth of the fungi, the cells are exposed to effective conditions to cause or allow the expression of the variant CBH2. In cases where a sequence encoding CBH2 is under the control of an inducible promoter, the inducing agent, for example, a sugar, metal salt or antibiotics, is added to the medium at a concentration effective to induce the expression of CBH2. In one embodiment, the strain comprises Aspergillus niger, which is a useful strain to obtain the overexpressed protein. For example, A. niger var awamori dgr246 is known to secrete high amounts of secreted cellulases (Goedegebuur et al, Curr. Genet (2002) 41: 89-98). Other strains of Aspergillus niger var to wamori such as GCDAP3, GCDAP4 and GAP3-4 are known Ward et al. (Ward, M, Wilson, LJ., And Kodama, K.H., 1993, Appl. Microbiol. Biotechnol., 39: 738-743). In yet another embodiment, the strain comprises Tri choderma reesei, which is a useful strain to obtain the over-expressed protein. For example, RL-P37, described by Sheri-Neiss et al., Appl. Microbiol. Biotechnol. 20: 46-53 (1984) is known to secrete high amounts of cellulase enzymes. Functional equivalents of RL-P37 include the strain RUT-C30 of Tri choderma reesei (ATCC No. 56765) and strain QM9414 (ATCC No. 26921). It is contemplated that these strains would also be useful in the overexpression of the CBH2 variant. Where it is desired to obtain the CBH2 variant in the absence of potentially harmful native cellulolytic activity, it is useful to obtain a host cell strain of Trichoderma that has had one or more cellulase genes deleted prior to the introduction of a cell culture construct.

DNA or plasma that contains the DNA fragment that codes for the CBH2 variant. Such strains can be prepared by the method described in U.S. Patent No. 5,246,853 and WO 92/06209, the descriptions of which are incorporated by reference herein. By expression of a variant CBH2 cellulase in a host microorganism that is lacking one or more cellulase genes, subsequent identification and purification procedures are simplified. Any gene from Trichoderma sp. which has been cloned can be deleted, for example, the cbhl, cbhl, egl and egl2 genes as well as those encoding the EGIII and / or EGV protein (see for example, U.S. Patent No. 5,475,101 and WO 94) / 28117, respectively). Deletion of the gene can be achieved by inserting a form of the desired gene that is to be deleted or disrupted in a plasmid, by methods known in the art. The deletion plasmid is then cut into one or several appropriate restriction enzyme sites, internal to the desired gene coding for the region, and the coding sequence of the gene or part thereof replaced with a selectable marker. Flanking DNA sequences from the locus of the gene to be deleted or disrupted, preferably between about 0.5 to 2.0 kb, remain on either side of the marker gene selectable An appropriate deletion plasmid will generally have the unique restriction enzyme sites present therein to make it possible for the fragment containing the deleted gene, including the flanking DNA sequences, and the selectable marker gene, to be eliminated as a single linear piece. . A selectable marker must be chosen to make possible the detection of the transformed microorganism. Any selectable marker gene that is expressed in the selected microorganism will be suitable. For example, with Aspergill us sp. , the selectable marker is chosen so that the presence of the selectable marker in the transformants will not significantly affect the properties thereof. Such a selectable marker can be a gene that codes for an assessable product. For example, a functional copy of a gene from Aspergill us sp. it can be used, which if absent in the host strain results in the host strain showing an auxotrophic phenotype. Similarly, there are selectable markers for Tri choderma sp. In one embodiment, a strain derived from pyrG 'of Aspergillus sp. is transformed with a functional pyrG gene, which thus provides a selectable marker for transformation. A strain derived from pyrG 'can be obtained by selection of strains of Aspergillus sp. which are resistant to fluoroorotic acid (FOA). The pyrG gene codes for orotidine-5'-monophosphate decarboxylase, an enzyme required for the biosynthesis of uridine. Strains with an intact pyrG gene develop in a medium that lacks uridine, but are sensitive to fluoroorotic acid. It is possible to select strains derived from pyrG 'that lack a functional orotidine-monophosphate-decarboxylase enzyme, and requires uridine for growth by selection for FOA resistance. Using the FOA selection technique it is also possible to obtain the strains that require uridine, which lack an orotate-pyrophosphoribosyl-transferase. It is possible to transform these cells with a functional copy of the gene that codes for this enzyme (Berges and Barreau, Curr. Genet 19: 359-365 (1991), and van Hartingsveldt et al., (1986) Development of a homologous transíormation system for Aspergillus niger based on the pyrG gene, Mol. Gen. Genet 206: 71-75). The selection of the derived strains is easily performed using the FOA resistance technique referred to above, and thus the pyrG gene is preferably employed as a selectable marker. In a second embodiment, a strain derived from pyr4 ~ of Hypocrea sp. (Hypocrea sp. (Trichoderma sp.)) Is transformed with a functional pyr4 gene, which in this way provides a selectable marker for transformation.

A strain derived from pyr4 'can be obtained by selection of strains of Hypocrea sp. (Trichoderma sp.) That are resistant to fluoroorotic acid (FOA). The pyr4 gene codes for orotidine-5'-monophosphate decarboxylase, an enzyme required for the biosynthesis of uridine. Strains with an intact pyr4 gene grow in a medium that lacks uridine, but are sensitive to fluoroorotic acid. It is possible to select strains derived from pyr4 ~ that lack a functional orotidine-monophosphate-decarboxylase enzyme, and require uridine for growth by selection for FOA resistance. Using the FOA selection technique it is also possible to obtain strains that require uridine lacking a functional orotate-pyrophosphoribosyl transferase. It is possible to transform these cells into a functional copy of the gene that codes for this enzyme (Berges &Barreau, 1991). The selection of the derived strains is easily performed using the FOA resistance technique referred to above, and thus, the pyr4 gene is preferably employed as a selectable marker. To transform pyrG 'Aspergill us sp. or pyr4 ~ Hypocrea sp. (Tri choderma sp.) So that they lack the ability to express one or more cellulase genes, a single DNA fragment comprising a disturbed or deleted cellulase gene, is then isolated from the suppression plasmid and used to transform an appropriate host of pyr-Aspergill us or pyr -Trichoderma. The transformants are then identified and selected based on their ability to express pyrG or pyr4, respectively, the gene product and this complements the uridin auxotrophy of the host strain. The Southern blot analysis is then carried out on the resulting transformants and confirming a double-crossing integration event that replaces part or all of the coding region of the genomic copy of the gene to be deleted with the pyr selectable markers. appropriate. Although the specific plasmid vectors described above refer to the preparation of the pyr ~ transformants, the present invention is not limited to these vectors. Several genes can be deleted and replaced in the strain of Aspergillus sp. or Hypocrea sp. (Tri choderma sp.) Using the previous techniques. In addition, any selectable, available markers may be used, as discussed above. In fact, any host, for example, Aspergill us sp. o Hyprocrea sp. , the gene that has been cloned, and thus identified, can be deleted from the genome using the strategy described above. As stated above, the host strains used can be derived from Hyprocrea sp. (Trichoderma sp.) That lack or that have a gene or genes not functionalities corresponding to the selectable marker chosen. For example, if the selectable marker of pyrG is chosen for Aspergillus sp. , then a strain derived from specific pyrG 'is used as a container in the transformation process. Also, for example, if the selectable marker of pyr4 is chosen for a Hyprocrea sp. , then a strain derived from specific pyr4 ~ is used as a container in the transformation process. Similarly, selectable markers comprising the genes of Hyprocrea sp. (Trichoderma sp.) Equivalent to the genes amdS, argB, trpC, niaD of Aspergill us nidulans, can be used. The corresponding container strain must therefore be a derivative strain such as argB ', trpG ~, niaD', respectively. The DNA encoding the CBH2 variant is then prepared for insertion into an appropriate microorganism. According to the present invention, the DNA encoding a variant of CBH2 comprises the DNA necessary to encode a protein having functional cellulolytic activity. The DNA fragment encoding the CBH2 variant can be functionally coupled to a fungal promoter sequence, for example, the glaA gene promoter in Aspergillus or the promoter of the cbhl or egl l genes in Tri choderma. It is also contemplated that more than one DNA copy which codes for a variant of CBH2 can be recombined within the strain to facilitate overexpression. The DNA encoding the variant CBH2 can be prepared by constructing an expression vector carrying the DNA encoding the variant. The expression vector carrying the inserted DNA fragment encoding the CBH2 variant can be any vector that is capable of autonomously replicating in a given host organism or integrating into the host DNA, typically a plasmid. In preferred embodiments, two types of expression vectors are contemplated to obtain expression of the genes. The first contains DNA sequences in which the promoter, the region encoding the gene, and the terminator sequence all originate from the gene to be expressed. Truncation of the gene can be obtained where desired by deletion of the unwanted DNA sequences (e.g., coding for unwanted domains) to leave the domain to be expressed under the control of its own transcriptional and translational regulatory sequences. . A selectable marker can also be contained on the vector allowing the selection for the integration within the host of multiple copies of the new gene sequences. The second type of expression vector is pre-assembled and contains sequences required for high-level transcription and a selectable marker. It is contemplated that the coding region for a gene or part thereof may be inserted into this expression vector for general purposes, such that it is under the transcriptional control of the promoter of the expression cassettes and the terminator sequences. For example, in Aspergillus, pRAX is such an expression vector for general purposes. The genes or part of them can be inserted downstream of the strong glaA promoter. For example, in Hypocrea, pTEX is such an expression vector for general purposes. The genes or part of them can be inserted downstream (3 ') of the strong cbhl promoter. In the vector, the DNA sequence encoding the CBH2 variant of the present invention must be operably linked to the transcriptional and translational sequences, for example, a suitable promoter sequence and the signal sequence in the reading structure to the genes structural The promoter can be any DNA sequence that shows transcriptional activity in the host cell and can be derived from the genes encoding the proteins either homologous or heterologous to the host cell. An optional signal peptide provides the intracellular production of the CBH2 variant. The DNA encoding the signal sequence is preferably that which is naturally associated with the gene to be expressed, however the signal sequence from any suitable source, for example an exo-cellobiohydrolase or Trichoderma endoglucanase, is contemplated in the present invention. The methods used to ligate the DNA sequences encoding the CBH2 variant of the present invention with the promoter, and the insertion into suitable vectors are well known in the art. The vector or DNA construct described above can be introduced into the host cell according to known techniques such as transformation, transfection, microinjection, microporation, biolistic bombardment and the like. In the preferred transformation technique, it should be taken into account that the permeability of the cell wall to DNA in Hypocrea sp. (Trichoderma sp.) Is very low. Consequently, the uptake of the desired DNA sequence, the gene or the gene fragment is at best minimal. There are a number of methods to increase the permeability of the cell wall of Hypocrea sp. (Tri choderma sp.) In the derived strain (for example, lacking a functional gene corresponding to the selectable marker used) before of the transformation process. The preferred method in the present invention for preparing Aspergillus sp. or Hypocrea sp. (Trichoderma sp.) For the transformation, involves the preparation of protoplasts from fungal mycelium. See Campbell et al. Improved transformation efficiency of A. niger using homologous niaD gene for nitrate reductase. Curr. Genet 16: 53-56; 1989. The mycelium can be obtained from germinated vegetative spores. The mycelium is treated with an enzyme that digests the cell wall, resulting in protoplasts. The protoplasts are then protected by the presence of an osmotic stabilizer in the suspension medium. These stabilizers include sorbitol, mannitol, potassium chloride, magnesium sulfate and the like. Usually, the concentration of these stabilizers varies between 0.8 M and 1.2 M. It is preferable to use approximately a 1.2 M solution of sorbitol in the suspension medium. DNA uptake within the host strain (Aspergillus sp. Or Hyprocrea sp. (Tri choderma sp.), Is dependent on the calcium ion concentration, generally between approximately 10 mM calcium chloride and 50 mM chloride. Calcium is used in an uptake solution In addition to the need for the calcium ion in the uptake solution, other generally included items are a buffer system such as a TE buffer (Tris 10 mM, pH 7.4; 1 mM EDTA) or 10 mM MOPS, buffer pH 6.0 (orfolinopropansulfonic acid) and polyethylene glycol (PEG). It is believed that polyethylene glycol acts to fuse cell membranes, thereby allowing the contents of the medium to be distributed to the cytoplasm of the host cell, for example by means of either the Aspergillus sp. Strain. or Hypocrea sp. , and the plasmid DNA transferred to the nucleus. This fusion often leaves multiple copies of plasmid DNA integrated into the host chromosome. Usually, a suspension containing the protoplasts or cells of Aspergillus sp. which have been subjected to a permeability treatment at a density of 105 to 106 / ml, preferably 2 x 105 / ml are used in the transformation. Similarly, a suspension containing the protoplasts of Hypocrea sp. (Tri choderma sp.) Or cells that have been subjected to a permeability treatment at a density of 108 to 109 / ml, preferably 2 x 108 / ml are used in the transformation. A volume of 100 μl of these protoplasts or cells in an appropriate solution (eg, 1.2 M sorbitol, 50 mM calcium chloride) are mixed with the desired DNA. In general, a high concentration of PEG is added to the uptake solution. 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplast solution. However, it is preferable add approximately 0.25 volumes to the protoplast suspension. The additives such as dimethyl sulfoxide, heparin, spermidine, potassium chloride and the like, can also be added to the uptake solution and aid in the transformation. In general, the mixture is then incubated at approximately 0CC for a period of between 10 to 30 minutes. Additional PEG is then added to the mixture to further increase the uptake of the desired gene or the desired DNA sequence. The 25% PEG 4000 is generally added in volumes of 5 to 15 times the volume of the transformation mixture; however, larger and smaller volumes may be suitable. The 25% PEG 4000 is preferably about 10 times the volume of the transformation mixture. After the PEG is added, the transformation mixture is then incubated either at room temperature or on ice before the addition of a solution of sorbitol and calcium chloride. The protoplast suspension is then further added to molten aliquots of a growth medium. This growth medium allows the growth of the transformants only. Any growth medium can be used in the present invention, which is suitable for growing the desired transformants. However, if the Pyr + transformants are being selected, it is It is preferable to use a growth medium that does not contain uridine. Subsequent colonies are transferred and purified on a growth medium depleted in uridine. In this stage, stable transformants can be distinguished from unstable transformants by their faster growth rate and, in Tri choderma, for example, the formation of circular colonies with a smooth rather than irregular contour in the solid culture medium that lacks uridine In addition, in some cases an additional stability test can be performed by growing the transformants on the non-selective solid medium (containing uridine), harvesting the spores of this culture medium and determining the percentage of these spores that will subsequently germinate and they will grow on the selective medium lacking uridine. In a particular embodiment of the above method, the CBH2 variant or variants are actively recovered from the host cell, after growth in liquid medium as a result of the appropriate post-translational processing of the CBH2 variant. (ii) Yeast The present invention also contemplates the use of yeast as a host cell for the production of CBH2. Other diverse genes that code for hydrolytic enzymes have been expressed in various strains of the yeast S. cerevisiae These include sequences coding for two endoglucanases (Penttila et al., Yeast vol.3, p 175-185, 1987) two cellobiohydrolases (Penttila et al., Gene, 63: 103-112, 1988) and a beta-glucosidase of Trichoderma reesei (Cummings and Fowler, Curr. Genet, 29: 227-233, 1996), a xylanase from Aureobasidi um pul l ulans (Li and Ljungdahl, Appl. Environ. Microbiol. 62, No. 1, pp. 209-213 , 1996), a wheat alpha-amylase (Rothstein et al., Gene 55: 353-356, 1987), etc. In addition, a cassette of the cellulase gene coding for the endo- [beta] -1,4-glucanase of Butyryvibrate fibrinosolve (END1), the cellobiohydrolase (CBH1) of Phanerochaete chrysosporium, the cellodextrinase (CEL1) of Ruminococcus flavefa hundreds and the celobiase (Bgll) from Endomyces fibrilli zer was successfully expressed in a laboratory strain of S. cerevisiae (Van Rensburg et al., Yeast, Vol 14, pp. 67-76, 1998). C. Introduction of Nucleic Acid Sequence Coding for CBH2 Within Host Cells The invention further provides cells and cell compositions that have been genetically modified to comprise a nucleic acid sequence encoding the variant CBH2, exogenously provided. A cell or progenitor cell line can be genetically modified (eg, transduced, transformed or transfected) with a cloning vector or a expression vector. The vector can be, for example, in the form of a plasmid, a viral particle, a phage, etc. as described further above. The transformation methods of the present invention can result in the stable integration of all or part of the transformation vector within the genome of the filamentous fungus. However, the transformation that results in the maintenance of a self-replicating extra-chromosomal transformation vector is also contemplated. Many standard transfection methods can be used to produce Tri choderma reesei cell lines that express large amounts of the heterologous protein. Some of the methods published for the introduction of DNA constructs within the Tri choderma cellulase producing strain include Lorito, Hayes, DiPietro and Harman, 1993, Curr. Genet 24: 349-356; Goldman, VanMontagu and Herrera-Estrella, 1990, Curr. Genet 17: 169-174; Penttila, Nevalainen, Ratto, Salminen and Knowles, 1987, Gene 6: 155-164, for Aspergill us Yelton, Hamer and Timberlake, 1984, Proc. Nati Acad. Sci. USA 81: 1470-1474, or Fusarium Bajar, Podila and Kolattukudy, 1991, Proc. Nati Acad. Sci. USA 88: 8202-8212, for Streptomyces Hopwood et al., 1985, The John Innes Foundation, Norwich, UK and for Bacill us Brigidi, DeRossi, Bertarini, Riccardi and Matteuzzi, 1990, FEMS Microbiol. Lett. 55: 135-138). Other methods for introducing a heterologous nucleic acid construct (expression vector) into filamentous fungi (eg, H. j ecorina) include, but are not limited to the use of a gene particle or gun, the permeabilization of cell walls of filamentous fungi before the transformation process (for example, by using high concentrations of alkali, for example, 0.05 M to 0.4 M of calcium chloride or lithium acetate), protoplast fusion or transformation mediated by Agrobacterium. An exemplary method for the transformation of filamentous fungi by treatment of protoplasts or spheroplasts with polyethylene glycol and calcium chloride is described in Campbell, E.I. et al., Curr. Genet 16: 53-56, 1989 and Penttila, M. et al., Gene, 63: 11-22, 1988. Any of the well-known methods for introducing foreign nucleotide sequences into host cells can be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, biolistics, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material within a host cell (see, for example, Sambrook et al., supra).

Also of use is the transfection method mediated by Agrobacterium um described in U.S. Patent No. 6,255,115. It is only necessary that the particular genetic engineering method used be able to successfully introduce at least one gene within the host cell capable of expressing the heterologous gene. In addition, heterologous nucleic acid constructs comprising a nucleic acid sequence encoding the CBH2 variant can be transcribed in vitro, and the resulting RNA introduced into the host cell by well known methods, for example, by injection. The invention further includes novel and useful filamentous fungal transformants such as H. jecorina and A. niger for use in the production of fungal cellulase compositions. The invention includes the filamentous fungal transformants, especially fungi comprising the sequence encoding the CBH2 variant, or the deletion of the sequence encoding endogenous cbh. After the introduction of a heterologous nucleic acid construct comprising the coding sequence for a variant of cbh2, the genetically modified cells can be cultured in conventional nutrient media, modified as appropriate to activate promoters, select transformants or amplify the expression of a nucleic acid sequence that codes for the CBH2 variant. The culture conditions, such as temperature, pH and the like, are those previously used for the host cell selected for expression, and will be apparent to those skilled in the art. The progeny of the cells into which such heterologous nucleic acid constructs have been introduced are generally considered to possess the nucleic acid sequence encoding the variant CBH2 found in the heterologous nucleic acid construct. The invention further includes novel and useful filamentous fungal transformants such as H. j ecorin for use in the production of fungal cellulase compositions. Aspergillus niger can also be used in the production of the CBH2 variant. The invention includes the filamentous fungal transformants especially fungi comprising the sequence encoding the cbhl variant, or the deletion of the sequence encoding endogenous cbh2. Stable transformants of filamentous fungi can be generally distinguished from unstable transformants by their faster growth rate and, in Tri choderma, for example, the formation of circular colonies with a smooth rather than irregular outline on the solid culture medium. In addition, in some cases an additional stability test may be performed by growth of the transformants on non-selective solid medium, harvesting the spores of this culture medium and determining the percentage of these spores that will subsequently germinate and develop on the selective medium. VII. Analysis for the Sequences Encoding for the CBH2 Nucleic Acid and / or Protein Expression In order to evaluate the expression of a variant of CBH2 by a cell line that has been transformed with a nucleic acid construct that codes for the variant of CBH2, tests can be carried out at the protein level, at the RNA level or through the use of functional bioassays, specific for the activity and / or production of cellobiohydralase. In an exemplary application of the nucleic acid of the CBH2 variant and the protein sequences described herein, a genetically modified strain of filamentous fungi, eg, Trichoderma reesei, is engineered to produce an increased amount of CBH2. Such genetically modified filamentous fungi could be useful to produce a cellulase product with increased cellulolytic capacity. In a procedure, this is accomplished by introducing the coding sequence for cM2 within a host cell, for example, a filamentous fungus such as Aspergillus niger. Accordingly, the invention includes methods for expressing the CBH2 variant in a filamentous fungus or other suitable host, by introducing an expression vector that contains the DNA sequence encoding the CBH2 variant within filamentous fungal cells. or other suitable hosts. In yet another aspect, the invention includes methods for modifying the expression of CBH2 in a filamentous fungus or other suitable host. Such modification includes a decrease or elimination in the expression of endogenous CBH2. In general, the assays used to analyze the expression of the CBH2 variant include, Northern blot, dot blot (DNA or RNA analysis), RT-PCR (reverse transcriptase polymerase chain reaction), or in-hybridization. if you, using an appropriately labeled probe (based on the nucleic acid coding sequence) and conventional Southern blot and autoradiography. In addition, the production and / or expression of the CBH2 variant can be measured in a sample directly, for example, by assays for activity, expression and / or cellobiohydrolase production. Such assays are described, for example, in Karlsson, J. et al. (2001), Eur. J. Biochem, 268, 6498-6507, Wood, T. (1988) in Methods in Enzymology, Vol. 160. Biomass Part a Cellulose and Hemicellulose (Wood, W. &Kellog, S. Eds. .), p. 19-25, Academic Press, San Diego, CA, USA) and, for the PAHBAH assay in (Lever, M. (1972) Analytical Biochemistry, 47, 273, Blakeney, AB &Mutton, LL (1980) Journal of Science of Food and Agriculture, 31, 889, Henry, RJ (1984) Journal of the Institute of Brewing, 90, 37. Useful substrates for evaluating the activities of cellobiohydrolase, endoglucanase or β-glucosidase include crystalline cellulose, filter paper, cellulose swollen with phosphoric acid, cellooligosaccharides, methylumbelliferyl-lactoside, methylumbelliferyl-cellobioside, orthonitrophenyl-lactoside, para-nitrophenyl-lactoside, orthonitrophenyl-cellobioside, para-nitrophenyl-cellobioside.In addition, the expression of the protein can be evaluated by immunological methods, such as immunohistochemical staining of The cells, titre sections or immunoassay of tissue culture medium, for example, by Western blotting or ELISA, such immunoassays can be used to assess qualitative and quantitatively the expression of a variant of CBH2. The details of such methods are known to those skilled in the art and many reagents to practice Such methods are commercially available. A purified form of a variant of CBH2 can be used to produce either monoclonal or polyclonal antibodies specific for the expressed protein, for use in various immunoassays. (See, for example, Hu et al., Mol Cell Biol, Vol 11, No. 11, p 5792-5799, 1991). Exemplary assays include ELISA, competitive immunoassays, radioimmunoassays, Western blotting, indirect immunofluorescent assays and the like. In general, commercially available antibodies and / or kits can be used for the quantitative immunoassay of the expression level of cellobiohydrolase proteins. VIII. Isolation and Purification of Recombinant CBH2 Protein In general, a variant CBH2 protein produced in cell culture is secreted into the medium, and can be purified or isolated, for example, by removing unwanted components from the cell culture medium. However, in some cases a variant CBH2 protein can be produced in a cellular form that needs recovery from a cell lysate. In such cases, the variant CBH2 protein is purified from the cells in which it was produced, using techniques routinely employed by those skilled in the art. The examples include, but are not limited to affinity chromatography (Tilbeurgh 'et al., FEBS Lett.16: 215, 1984), ion exchange chromatographic methods (Goyal et al., Bioresource Technol. 36: 37-50, 1991; Fliess. et al., Eur. J. Appl. Microbiol. Biotechnol., 17: 314-318, 1983; Bhikhabhai et al., J. Appl. Biochem. 6: 336-345, 1984; Ellouz et al., J. Chromatography 396 : 307-317, 1987), including ion exchange using materials with high resolving power (Medve et al., J. Chromatography A 808: 153-165, 1998), hydrophobic interaction chromatography (Tomaz and Queiroz, J. Chromatography A 865: 123-128, 1999), and two-phase division (Brumbauer, et al., Bioseparation 7: 287-295, 1999). Typically, the variant CBH2 protein is fractionated to secrete proteins having selected properties, such as binding affinity to particular binding agents, eg, antibodies or receptors; or having a selected molecular weight range, or range of isoelectric points. Once expression of a given variant CBH2 protein is achieved, the produced CBH2 protein is purified from the cells or from the cell culture. Exemplary methods suitable for such purification include the following: antibody affinity column chromatography, ion exchange chromatography; precipitation with ethanol; Reverse phase HPLC; chromatography on silica or on a cation exchange resin such as DEAE; chromatofocusing; SDS-PAGE; precipitation with ammonium sulfate; and gel filtration using, for example, Sepahdex G-75. Various methods of protein purification can be employed and such methods are known in the art and described for example in Deutscher, Methods in Enzymology, Vol. 182, No. 57, p. 779, 1990; Scopes, Methods Enzymol. 90: 479-91, 1982. The selected purification step (s) will depend, for example, on the nature of the production process used and the particular protein produced. IX. Utility of cbhl and CBH2 It can be appreciated that the cbhl variant nucleic acids, the variant CBH2 protein and the compositions comprising the variant CBH2 protein activity find utility in wide variety applications, some of which are described below. . New and improved cellulase compositions comprising varying amounts of cellulases type BG, type EG and variant type CBH, find utility in detergent compositions that exhibit improved cleaning ability, function as a softening agent and / or improve the feel to the cotton fabrics (for example, "stonewashing" or "bio-polishing"), in compositions for degrading wood pulp into sugars (for example, for production of bio-ethanol), and / or in food compositions. The isolation and characterization of the cellulase of each type provides the ability to control the aspects of such compositions. Variable (or mutant) CBHs with increased thermostability find uses in all of the above areas due to their ability to retain activity at elevated temperatures. Variant (or mutant) CBHs with increased thermostability find uses, for example, in areas where enzymatic activity requires that the enzymatic activity be neutralized at lower temperatures, so that other enzymes that may be present are left unaffected. In addition, enzymes can find utility in the limited conversion of cellulosics, for example, in the control of the degree of crystallinity or the length of cellulose chain. After reaching the desired degree of conversion, the saccharification temperature can be raised above the survival temperature of destabilized CBH. Since the activity of CBH is essential for the hydrolysis of crystalline cellulose, the conversion of crystalline cellulose will cease at the elevated temperature. In one method, the cellulase of the invention finds utility in detergent compositions or in the Treatment of fabrics to improve the feeling to the touch and appearance. Since the rate of hydrolysis of cellulosic products can be increased by the use of a transformant having at least one additional copy of the cbh gene inserted into the genome, products containing cellulose or heteroglycans can be degraded at a faster rate and a greater degree. Processed cellulose products such as paper, cotton, cellulose diapers and the like can be degraded more efficiently in a sanitary landfill. In this way, the fermentation product obtainable from the transformants or the transformants alone can be used in compositions to help degrade by liquefaction, a variety of cellulose products that are added to the saturated landfills. Separate saccharification and fermentation is a process by which the cellulose present in the biomass, for example, corn cobs, is converted to glucose and subsequently the yeast strains convert the glucose into ethanol. Simultaneous saccharification and fermentation is a process by which the cellulose present in the biomass, for example, corn cobs, is converted to glucose and, at the same time and in the same reactor, the yeast strains convert the glucose into ethanol . In this way, In yet another method, the variant CBH cellulase of the invention finds utility in the degradation of biomass to ethanol. The production of ethanol from readily available sources of cellulose provides a stable, renewable fuel source. Cellulose-based feed materials are comprised of agricultural waste, pasture and wood and other low-value biomass such as municipal waste (eg, recycled paper, yard trimmings, etc.). Ethanol can be produced from the fermentation of any of these cellulosic feedstocks. However, cellulose must first be converted to sugars before there can be any conversion to ethanol. A wide variety of feed materials may be used with the variant CBH of the invention, and that selected for use may depend on the region where the conversion is being made. For example, in the Midwest of the United States, agricultural waste such as wheat straw, corn cobs and bagasse may predominate, while rice straw may predominate in California. However, it should be understood that any available cellulosic biomass can be used in any region. A cellulase composition containing a Increased amount of cellobiohydrolase, finds utility in the production of ethanol. Ethanol from this process can also be used as an octane booster or directly as a fuel instead of gasoline, which is advantageous because ethanol as a fuel source is more environmentally friendly than petroleum products . It is known that the use of ethanol will improve air quality and possibly reduce local ozone and smoke levels. In addition, the use of ethanol instead of gasoline may be of strategic importance in buffering the impact of sudden changes in non-renewable energy and petrochemical supplies. Ethanol can be produced via saccharification and fermentation processes from cellulosic biomass such as trees, herbaceous plants, municipal solid waste and agricultural and forestry residues. However, the proportion of individual cellulase enzymes within a mixture of cellulases of natural origin produced by a microbe may not be the most efficient for the rapid conversion of cellulose into biomass to glucose. It is known that endoglucanases act to produce new ends of cellulose chain that by themselves are substrates for the action of cellobiohydrolases and with this improves the efficiency of the hydrolysis of the complete cellulase system. Thus, the use of increased or optimized activity of cellobiohydrolase can greatly increase ethanol production. Thus, the cellobiohydrolase of the invention finds use in the hydrolysis of cellulose to its sugar components. In one embodiment, a variant cellobiohydrolase is added to the biomass before the acquisition of a fermentative organism. In a second embodiment, a variant cellobiohydrolase is added to the biomass at the same time as a fermentative organism. Optionally, other cellulase components present in any mode may exist. In yet another embodiment, the cellulosic feedstock can be pre-treated. The pre-treatment can be by high temperature and the addition of either dilute acid, concentrated acid or dilute alkali solution. The pre-treatment solution is added for a sufficient time to at least partially hydrolyze the hemicellulose components, and then neutralized. The major product of the action of CBH2 on cellulose is cellobiose, which is available for conversion to glucose by BG activity (for example in a fungal cellulase product). Either by pre-treating the cellulosic mass or by enzymatic action on the biomass, other sugars, in addition to the glucose and cellobiose, can be made available from biomass. The hemicellulose content of the biomass can be converted (by the hemicellulases) to sugars such as xylose, galactose, mannose and arabinose. Thus, in a biomass conversion process, enzymatic saccharification can produce sugars that are made available for biological or chemical conversions to other intermediates or end products. Therefore, sugars generated from biomass find use in a variety of processes in addition to the generation of ethanol. Examples of such conversions are the fermentation of glucose to ethanol (as reviewed by ME Himmel et al., Pp. 2-45, in "Fuels and Chemicals from Biomass", ACS Symposium Series 666, ed. BC Saha and J. Woodward , 1997) and other biological conversions of glucose to 2,5-diketo-D-gluconate (U.S. Patent No. 6,599,722), lactic acid (R. Datta and SP.Tsai p.222-436, ibid), succinate (RR Gokarn, MA Eiteman and J. Sridhar P. 237-263, ibid), 1,3-propanediol (AP Zheng, H. Biebl and WD Deckwer P. 264-279, ibid), 2,3-butanediol (CS Gong, N. Cao and GT, Tsao p.280-293, ibid) and the chemical and biological conversions of xylose to xylitol (BC Saha and RJ Bothast pp307-319, ibid). See also, for example, WO 98/21339. The detergent compositions of this invention can employ in addition to the cellulase composition (regardless of the cellobiohydrolase content, eg, free of cellobiohydrolase, substantially free of cellobiohydrolase or increased cellobiohydrolase), a surfactant, including anionic, nonionic and ampholytic surfactants, a hydrolase, additive agents, bleaching agents, bluing agents and fluorescent dyes, cake formation inhibitors, solubilizers, cationic surfactants and the like. All these components are known in the detergent art. The cellulase composition as described above can be added to the detergent composition either in a liquid diluent, in granules, in emulsions, in gels, in pastes and the like. Such forms are well known to the person skilled in the art. When a solid detergent composition is employed, the cellulase composition is preferably formulated as granules. Preferably, the granules can be formulated to thus contain a cellulase protective agent. For a more complete discussion, see U.S. Patent No. 6,162,782 entitled "Detergent compositions containing cellulase compositions deficient in CBH2 type components", which is incorporated by reference herein. Preferably, the cellulase compositions are employed from about 0.00005 weight percent to about 5 weight percent relative to the Total detergent composition. More preferably, the cellulase compositions are employed from about 0.0002 weight percent to about 2 weight percent relative to the total detergent composition. In addition, the nucleic acid sequence of the CBH2 variant finds utility in the identification and characterization of related nucleic acid sequences. A number of useful techniques for determining (predicting or confirming) the function of related genes or gene products include, but are not limited to, (A) DNA / RNA analysis, such as (1) overexpression, ectopic expression, and expression in other species; (2) deletion of a gene (reverse genetics, targeted deletion of a gene, gene silencing induced by viruses (VIGS, see Baulcombe, 100 Years of Virology, Calisher and Horzinek eds., Springer-Verlag, New York, NY 15: 189-201, 1999), (3) analysis of the state of mutilation of the gene, especially the flanking regulatory regions, and (4) hybridization in itself, (B) analysis of the gene product such as (1) the expression of the protein recombinant; (2) production of antisera; (3) immunolocalization; (4) biochemical assays for catalytic activity or other activity; (5) phosphorylation status; (6) interaction with other proteins via the analysis of two yeast hybrids; C) analysis of the pathway, such as the placement of a gene or gene product within a particular biochemical or signaling pathway, based on its overexpression phenotype or by sequential homology with related genes; and (D) other analyzes that can also be performed to determine or confirm the participation of the isolated gene and its product in a particular metabolic or signaling pathway, and help determine the function of the gene. All patents, patent applications, articles and publications mentioned herein are expressly incorporated by reference herein. EXAMPLES The present invention is described in further detail in the following examples, which are in no way intended to limit the scope of the invention as claimed. It is understood that the appended Figures are considered as integral parts of the specification and description of the invention. All references cited herein are specifically incorporated by reference for all that is described herein. EXAMPLE 1 Alignment of known CelßA cellulases The choice of several of the mutations was determined primarily by the alignment of Cel6A from Hypocrea j ecorina to eight (8) family members, using structural information and a program of modeling Figure 3 shows the alignment of the CBH2 molecules derived from Humicola insolens (Q9C1S9), Acremonium cell ulotyti cus (093837), Agaricus bispor? S (P49075), Hypocrea koningii (AF315681), Phanerochaete chrysosporium (S76141), Talaromyces emersonii (Q8N1B5), Len tinula edodes (AF244369), Hypocrea j ecorina (P07987). The alignments were made using Clustal W with an empty space penalty of 10 using the Vector NTI Suite software program. Based on the alignments, various mutations of single and multiple amino acids were made in the protein by site-directed mutagenesis. Possible mutations were identified, which can improve the thermostability of the enzyme by using the consensus sequence. See Figure 3. A visual inspection of the three-dimensional structure was performed to verify its compatibility with the structure. All changes that do not fit within the CBH2 molecule for spherical reasons or are close to the active site were omitted from the group of initial mutations. The consensus sequence for CBH2 was determined by the alignment of the CBH2 sequences as described herein. The alignment of Figure 3 served as the basis for the determination of the so-called consensus sequence. The consensus sequence of an alignment is the sequence, which has in each position the amino acid that is found in most of the amino acid sequences, which were used to construct the alignment. Those positions where the consensus sequence deviated from the CBH2 amino acid sequence of T. reesei were evaluated by examining the three-dimensional structure of the protein (PDQ code 1QK2). Graphic inspection was performed using the BRAGI computer program (D. Schomburg, J. Reichelt, J. Mol. Graphics, Vol.6, 161-165 (1988)) on a Silicon Graphics Indigo2 Solid Impact computer. Those mutations that -according to the three-dimensional model- fit in the structure without perturbation and probably improved the thermostability of the enzyme, were selected as the replacement for the improved thermostability of H. j ecorina CBH2. In some cases, visual inspection of the three-dimensional structure of the CBH2 molecule made it necessary to replace a non-conserved residue of the H. j ecorina CBH2, by another amino acid that the sequential alignment suggested. In some cases, the amino acid is glycosylated in the three-dimensional structure. The glycosylated positions, which were investigated based on the alignment, are S109 and N310. These positions were not changed. In position V94 the valine was replaced by a glutamic acid residue, because it can have stabilizing charge interactions with one or two arginines in position 213 and / or 237. In position T142 it was decided to introduce a proline, which can fit according to the alignment of the sequence. This amino acid is found in many of the aligned sequences and can stabilize due to the entropic effect of proline. At position L179 it was decided to test the effect of an introduction of an alanine, which is the only alternative amino acid in the CBH2 molecules in this alignment. At position Q204, it was decided to replace glutamine with a glutamic acid residue and not with alanine, as suggested by the consensus sequence, because the introduction of alanine can destroy favorable interactions in the hydrophobic core, while the introduction of a charge through the mutation Q to E must improve the charge network on the surface of the molecule. V206 was replaced by leucine, because the fit within the hydrophobic core appears better than the isoleucine setting. In the case of V250, it was decided to replace it with leucine and not with the slightly larger isoleucine due to spatial constraints. In position N285, the influence of the length of the side chain on stability was investigated, by replacing asparagine with glutamine. In position S291, it was decided to test the effect of an introduction of a glycine, which is the only alternative amino acid in the CBH2 molecules in this alignment. In position S316 it was decided to test the effect of an introduction of a proline, which is the only alternative amino acid in the CBH2 molecules in this alignment. In position S343, it was decided to introduce a proline due to its stabilizing effect on the main chain, and the fact that this amino acid is not the most frequent in this position, in the alignment. At position T349 threonine was replaced by a leucine and not valine as suggested by the consensus sequence. In position S413 it was decided to test the effect of an aromatic residue on stability in this position, by replacing serine with tyrosine. EXAMPLE 2 Preparation of the cbh2 constructs The cDNA sequence of CBH2 presented in the Figure 2 served as the template for the gene amplification. This also served as the template for the introduction of mutations. The following DNA primers were constructed for use in the amplification of cbh2 mutant genes from the genomic DNAs isolated from various microorganisms. All symbols used herein for the protein and DNA sequences correspond to the codes of the IUPAC IUB Biochemical Nomenclature Commission.

The primers 5 'FRG361) and 3' (FRG362) were developed based on the sequence of c 2 of Trichoderma reesei. Both primers contained the Invitrogen® Entry cloning sequences at the 5 'end of the primer. Primer 361 contained the attBl sequence and primer FRG362 contained the attB2 sequence. Sequence of FRG361 without attBl: ATGATTGTCG < XATTCTCAC (this primes the 5 'end of the gene, which codes for the CBH2 signal sequence of H. jecorina) (SEQ ID NO: 3) Sequence of FRG362 without attB2: TTACAGGAACGATGGGTTTGCG (this primes the 3' end of the gene that encodes for the catalytic domain of H. jecorina CBH2) (SEQ ID NO: 4)). The cbhl cDNA of H. jecorina served as a template. The cDNA used was derived from a cDNA library prepared as described by Pamela K. Foreman et al, Journal of Biological Chemistry Vol. 278, No. 34 2003 page 31989. The library was selected with the probe of the catalytic domain of CBH2. using the primers: Table 1 The PCR conditions were as follows: 10 μl of 10X reaction buffer (the 10X reaction buffer comprising 100 mM Tris HCl, pH 8-8.5, 250 mM potassium chloride, 50 mM ammonium sulfate, 20 mM magnesium sulfate); 0.2 mM of each of dATP, dTTP, dGTP, dCTP (final concentration), 1 μl of DNA at 100 ng / μl, 0.5 μl of PWO polymerase (Boehringer Mannheim, Cat # 1644-947) at 1 unit per μl, 0.2 μM of each primer, FRG361 and FRG362 (final concentration), 4 μl of DMSO and water up to 100 μl. The fragments coding for the variants were purified from a randomose gel using the Qiagen Gel extraction equipment. The purified fragments were used to perform a clone reaction with the vector pDONRMR 201 vector from Invitrogen®, using the Gateway ™ Technology manual (version C) from Invitrogen®, incorporated by reference herein. The pENTRYCBH2 clone prepared in this way is given in the Figure. The variant sites in CBH2 of H. jecorina may be involved in the thermostability of the variants and the cbhl gene of H. j ecorina was therefore subjected to mutagenesis using the primers and reaction reagents described below. The cyclization parameters for each reaction (single-site mutagenesis, random mutagenesis, regional or combinatorial mutagenesis) were the same: 1 Table 2 The amplification products were isolated and characterized as described below (see Examples 3-6). Genes (variants or wild-type) having the correct sequence were then transferred from the ENTRY vector to the target vector (pRAXdes2) to obtain the expression vector pRAXdesCBH2. The cells were transformed with an expression vector comprising a nucleic acid encoding the variant CBH2 cellulase. The constructions were transformed into A. niger variety awamori according to the method described by Cao et al. (Cao QN, Stubbs M, Ngo KQP, Ward M, Cunningham A, Pai EF, Tu GC and Hofmann T (2000) Penicillopepsin-JT2 to recombinant enzyme from Penicillium janthinellum and contribution to a hydrogen bond in subsite S3 to kcat Protein Science 9 : 991-1001). EXAMPLE 3 Mutagenesis and rigid site Based on the previous explanations presented in the 1 Example 1, site-directed CBH2 mutants were made with the following 5'-phosphorylated primers that were developed and synthesized using techniques well known in the art. Table 3: Primers for Mutants Directed to a Unique Site The codons that code for the mutation are underlined and in bold. In order to develop the Mutants Directed to the Site, the Mutagenesis team directed to the QuikChange Multi Site-Directed Mutagenesis Kit site (Stratagene, La Jolla, CA; Cat. # 200513) was used. The mutagenesis reaction was carried out using the following reaction reagents: TABLE 4 The amplification products were recovered (purified from the primers, nucleotides, enzymes, mineral oil, salts and other impurities) were digested with Dpnl. One microliter of DpnI (10 U / μL) was added to the PCR mixture and incubated at 37 ° C for 90 minutes. The PCR reaction products were purified using the QIAquick PCR Purification Kit (250) (Qiagen, Cat. No. 28106) according to the manufacturer's instructions. The elution volume was 35 μl of elution buffer. To remove the double-stranded non-mutated DNA, the eluted sample was digested a second time with the restriction enzyme DpnI. 1 μl of Dpnl from Invitrogen (Cat. No. 15242-019) and 4 μl of the reaction buffer (Reagent 4 is supplied with Dpnl as a 10X solution, dilute 1:10 in the final reaction mixture) were added to the sample and incubated at 37 ° C for 90 minutes. Two microliters of the reaction mixture were used for the transformation of 10 μl of the electro-competent cells One-Shot ToplO (Invitrogen, Cat. No. C4040-50). After 1 hour of growth at 37 ° C in the SOC medium (See Hanahan (1983) J.

Mol. Biol. 166: 557-580), the cells were seeded on selective kanamycin plates, and incubated at 37 ° C overnight. Positive clones were developed in the 2 * TY medium with 100 μg / ml ampicillin, and the plasmid DNA was isolated with the QIAprep Spin Miniprep Kit (Cat. No. 27106). The plasmids were sequenced to confirm that the Mutation sequence had been incorporated correctly. Mutants with the correct sequence were transferred to the expression vector of A. n i ger pRAXdest # 2 with the LR reaction, according to the Gateway Cloning procedure (Invitrogen, Cat. No. 11791019). After transformation of protoplasts from the expression clones to A. n i ger AP4, site-directed mutants (SDM) were selected for altered thermostability (Table 14). EXAMPLE 4 Combinatorial Libraries Two QuickChange libraries (QC2C and QC2D) were developed, based on the results for the single site mutations that were identified during the SDM selection: 98, 134, 206, 212, 312, 316, 411 and 413. Mutations P98L, M134V, V206L, 1212V, T312S, S316P, F411Y and S413Y were randomly combined in libraries using the quick-change (QC) method. To develop the QC libraries, the Multi-Site Mutagenesis Team was used (Cat # 200513 from Stratagene). The primers were prepared as listed in the following table: Table 5 A mixture of primers was prepared as follows: Thirty microliters of primers 98 and 134 (see table above) were mixed with 10 μl of each other primer (see table above). Two different concentrations of primers were tested resulting in two libraries of which one contained an average of 2 amino acid substitutions and the second 6 amino acid substitutions per molecule. The mutagenesis reaction was carried out using the following reaction reagents: Table 6 The amplification products were digested with Dpnl. One microliter of DpnI (10 U / μL) was added to the PCR mixture and incubated at 37 ° C for 90 minutes. The PCR reaction products were purified using the QIAquick PCR Purification Kit (250) (Qiagen, Cat. No. 28106) according to the manufacturer's instructions. The elution volume was 35 μl elus ion buffer. To remove the double-stranded non-mutated DNA, the eluted sample was digested a second time with the restriction enzyme DpnI. 1 μl of DpnI from Invitrogen (Cat. No. 15242-019) and 4 μl of the reaction buffer (Reagent 4 is supplied with Dpnl as a 10X solution, dilute 1:10 in the final reaction mixture) were added to the sample and incubated at 37 ° C for 90 minutes. Two microliters of the reaction mixture were used for the transformation of 10 μl of the One-Shot ToplO electro-competent cells (Invitrogen, Cat. No. C4040-50). After 1 hour of growth at 37 ° C in the SOC medium (See Hanahan (1983) J. Mol. Biol. 166: 557-580), the cells were seeded on selective kanamycin plates, and incubated at 37 ° C. all night. Positive clones were developed in the 2 * TY medium with 100 μg / ml ampicillin, and the plasmid DNA was isolated with the QIAprep Spin Miniprep Kit (Cat. No. 27106). The plasmids were sequenced to confirm that the mutation sequence had been correctly incorporated. Mutants with the correct sequence were transferred to the expression vector of A. n i ger pRAXdest # 2 with the LR reaction, according to the Gateway Cloning procedure (Invitrogen, Cat. No. 11791019). After transformation of protoplasts from the expression clones to A. n i ge r AP4, the QC libraries were selected for the thermostability (Tables 15, 16). EXAMPLE 5 Regional Mutagenesis As described below, based on the results for the single-site mutations that were identified during the selection of SDM, the regions were identified in the three-dimensional structure of CBH2, randomly mutated and classified in a thermostability assay. . The amino acids, which constitute such a spatial region, are (in groups): [210, 214], [253, 255, 257, 258], [411, 413, 415], [412, 414, 416], [312,313] , 323, [212, 149, 152], [134, 144] and 98. Completely randomized libraries in the previous positions (for example, [210, 214], [253, 255, 257, 258] , [411, 413, 415], [412, 414, 416], [312,313], 323, [212, 149, 152], [134, 144] and 98) were selected. The amino acids in the above list in brackets (for example, [210, 214]) were read together, amino acids 323 and 98 were alloyed alone. The CBH2-S316P or CBH2-V206L-S316P variants served as the main chain for these libraries. The NNS primers were constructed and ordered from Invitrogen: Table 7 The PCR was carried out according to the following protocol, with the Pfu Ultra DNA polymerase (Stratagene, Cat. No. 600380): Table 8 The PCR fragments were placed in a 1% LMT gel, and purified with a Qiagen Gel Extraction Kit (Stratagene Cat. No. 28706). The purified fragments were fused with Pfu Ultra (see above) and the cbhl primers with the flanking attB sequences. The purified ci 2 genes were transferred to a Gateway Entry-Vector pDON201 with the BP reaction, according to the manual (Invitrogen, Cat. No. 11789013). The positive clones were selected on 2 * TY plates with kanamycin (50 μg / ml), and the plates were scraped for the preparation of the plasmids for transfer to pRAXdes # 2 (expression vector), with the Gateway LR reaction ( Invitrogen; Cat. No. 11791019). This vector was digested with Notl, to optimize the frequency of transformation of the reaction of LR. The transformation of protoplasts has been used to create 9 Regional Libraries of CBH2 in A. niger AP4 that were selected for altered thermostability (Table 17). EXAMPLE 6 Multiple Mutants Based on the expression and thermostability results of the single-site mutations, a group of multiple mutants was designed, which were produced in shake flasks only. Mutations of mutant CBH2 FCA557 (P98L / M134V / S316P / S413Y) (from the QC libraries, Example 4) were combined (Table 9) with those of CBH2 mutants FCA 564 (S316P / V323Y), FCA568 (V206L / S210R / T214Y / S316P) and FCA570 (M134L / L144R / S316P) (from the Regional Libraries, Example 5), to obtain CBH2 molecules with improved thermal stability. ti H • 3 THE THE The primers were constructed and ordered from Invitrogen: Table 10 PCR was performed using the reaction reagents in Table 11 (below), to obtain all the fragments (A-M) necessary for build the 9 combineries of CBH2. Phusion DNA polymerase (Finnzymes, Cat. No. F-530) was used. The primer concentration was 10 μM. Cycle conditions are given in Table 2 (above). Table 11 The PCR fragments were placed on a 1% LMT gel, and purified with a Qiagen Extraction Kit (Stratagene Cat. No. 28706). The purified fragments were fused using the Phusion DNA polymerase (see above) with the CBH2-attB primers (above), to obtain the combinatorics of complete CBH2 according to Table 12. Table 12 The complete CBH2-attB molecules were purified from 1% LMT agarose and transferred to the expression vector pRAXdes # 2 of A. niger AP. The method used was the "Protocol of a tube for the cloning of attB-PCR products directly into target vectors" (according to the Invitrogen manual). Three microliters of the reaction sample were used for the transformation of 100 μl of competent cells of maximum efficiency DH5a (Invitrogen Cat. No. 18258012), according to the manual. After 1 hour of growth at 37 ° C in SOC medium, the cells were seeded on selective ampicillin plates (100 μg / ml), and incubated at 37 ° C overnight. Positive clones were developed in 2 * TY medium and 100 μg / ml ampycillin. He Plasmid DNA was isolated with the QIAprep Spin Kit Miniprep Kit (Qiagen Cat. No. 27106) and sequenced. The mutants with the correct sequence were transferred to the expression vector pRAXdes # 2 of A. niger with the LR reaction, according to the Gateway Cloning procedure (Invitrogen; Cat. No. 11791019). After transformation of protoplasts from the expression clones to A. niger AP4, Multiple Mutants were expressed and isolated (as in Example 7), and analyzed for thermostability (Tables 18, 19). EXAMPLE 7 Expression and Isolation of CBH2 and its variants from shake flask growths To provide the materials for the Tm measurements in the thermal denaturation studies (Example 9), the expression clones were developed in shake flasks and then the CBH2 molecules were purified, as follows: The cells were transformed with an expression vector comprising a nucleic acid encoding the variant CBH2 cellulase. The constructions were transformed inside A. n i ger variety to wamori according to the method described by Cao et al. (Cao QN, Stubbs M, Ngo KQP, Ward M, Cunningham A, Pai EF, Tu GC and Hofmann T (2000) Penicillopepsin-JT2 to recombinant enzyme from Penicillium janthinellum and contribution to a hydrogen bond in subsite S3 to kcat Protein Science 9 : 991-1001). The transformants of A. n i ger variety to wamori were developed on minimum medium that lacked uridine (Ballance et al., 1983). Transformants were developed by inoculating 1 cm2 of spore suspension from the sporulated developed agar plate, in 100 ml shake flasks for 3 days at 37 ° C as described by Cao et al. (2000), and then selected for cellulase activity. The CBH2 activity assay was based on the hydrolysis of Phosphoric Acid Swollen Cellulose (PASC: 0.5% PASC in 0.5 mM sodium acetate, pH 4.85). The reducing sugars were measured by the test of PAHBAH. (PAHBAH: 2975 g of PAHBAH, 9.75 g of sodium and potassium tartrate in 195 ml of 2% sodium hydroxide). PASC: (Karlsson, J. et al. (2001), Eur. J. Biochem, 268, 6498-6507, Wood, T. (1988) in Methods in Enzymology, Vol. 160. Biomass Part a Cellulose and Hemicellulose (Wood , W. &Kellog, S. Eds.), Pp. 19-25, Academic Press, San Diego, CA, USA) and PAHBAH: (Lever, M. (1972) Analytical Biochemistry, 47, 273, Blakeney, AB &Mutton, LL (1980) Journal of Science of Food and Agriculture, 31, 889, Henry, RJ (1984) Journal of the Institute of Brewing, 90, 37). The variants and the wild type of CellA were then purified from the cell-free supernatants of these cultures by hydrophobic interaction chromatography (HIC) by one of two methods: For the SDM variants (Example 3), Poly-Bio-RAD Columns Prep CAT # 731-1550 were used by Pharmacia phenyl sepharose resin (1.6 ml = 1 ml column) CAT # 17-0973-05. The resin was allowed to settle before washing with 1 to 2 volumes of column water (CV), then it was balanced with 5 CV of sodium phosphate 0. 020 M, 0.5 M ammonium sulfate, pH 6.8 (Shock absorber TO) . 4 M ammonium sulfate was added to the supernatants to a final concentration of 0.5 M. 2 CV of the supernatant were loaded and the column was washed with 5 CV of Buffer A, before elution with 4 CV of 0.020 M sodium phosphate, pH 6.8. The filtrate contained purified CBH2. For Multiple Mutants (Example 6), the columns were run on a Novagen vacuum pipe using the Poros® 20 HP2 resin made by Applied Biosystems. The HIC columns were equilibrated with 5 CV of Buffer A. Ammonium sulfate was added to the supernatants to a final concentration of approximately 0.5 M and the pH was adjusted to 6.8. After filtration, the supernatant was loaded, the column washed with 10 C of Buffer A and then eluted with 10 CV of 0.020 M sodium phosphate, pH 6.80. Fractions were collected and combined based on the presence of CBH2, as detected by reduced SDS-PAGE gel analysis. When desired, the CBH2 molecules are deglycosylated before purification by treatment of the supernatant with Endoglucosidase H according to the protocol provided (Sigma-Aldrich). EXAMPLE 8 Thermostability of CBH2 variants by thermal inactivation CBH2 molecules with altered stability to irreversible thermal inactivation compared to wild-type CBH2, were identified by measuring the PASC activity of equal aliquots of cell-free supernatants, before and after incubation at elevated temperature under stringent conditions. The stringent conditions used were incubation for 1 hour at either 61 ° C or 65 ° C (as indicated) of a 1: 1 dilution of the supernatant in 0.1M sodium acetate, pH 4.85, followed by ice cooling for 10 minutes. minutes Percent residual activity (% of remaining activity after incubation at elevated conditions) was calculated by dividing the residual activity by the initial activity (CBH2 activity on PASC before severe incubation). The selection of CBH2 and wild-type variants for thermal inactivation stability was carried out according to the following protocols: A. Solutions and media The following solutions / media were used in the determination of the stability of the mutants of CBH2 to irreversible thermal inactivation: 1. Minimum medium plus maltose (MM medium) was prepared as shown in Table 13: Table 13: Minimum medium + maltose for A. Niger Elements in -1 g FeS04 «7H20 traces-LW (L. -8.8 g ZnS0» 7H20 Wilson) -0.4 g CuS04 »5H20 -0.15 g MnS04 »4H20 -0.1 g Na2B4O7 »10H2O -50 mg [NH4) 6M? 7024 »4H20 -250 ml distilled water, stir and add -0.20 ml concentrated HCl to dissolve the crystals (4N) Rinse to 1 liter with distilled water and sterilize by filtration (0.2 μm) K-cushion ? HP04 1 M Phosphate pH 5.8 KH2P04 1 M Mix both solutions until the pH = 5.8 and sterilize by filtration (0.2 μm) 2. 0.5% PASC solution in 50 mM sodium acetate, pH 4.85: i. Add 5 grams of Avicel PH101 (Fluka 11365) to a 1 liter glass flask and add approximately 12 ml of water to make a thick suspension. ii. Place the flask on ice. iii. Add 150 ml of 85% ortho-phosphoric acid cooled with ice (Art. 1000573 Merck) and mix with the ultra turrax at high speed (prevent splashing) for approximately 1 hour. iv. Add 100 ml of ice-cold acetone, which causes a slow precipitation of the amorphous cellulose to a very thick suspension. Use a spatula to mix better. v. Dilute the very thick suspension to about 1 liter with water to make it fluid enough to transfer it to 6 Sorvall 250 ml containers. saw. Centrifuge 15 minutes at 10k and discard the supernatant. vii. Mix the pellets with as much water as the containers can hold, and centrifuge again. viii. Repeat steps 5 and 6 at least 3 times until the pH has increased to pH 4.0-5.0. ix. To increase the phosphoric acid wash, a drop of 4N sodium hydroxide can be added to the water. x. Mix the last pellets with water until approximately 300 ml and homogenize. xi. Determine the concentration of the suspension with dry weight measurement.

Sterilize the suspension for 20 minutes at 121 ° C. Cool and store in the refrigerator. 3. The PAHBAH reagents were prepared as follows: 1.5 g of PAHBAH + 5 g of sodium and potassium tartrate in 100 ml of 2% sodium hydroxide 4. Cellobiose stock solutions; in MQ water prepare a solution at 0.01, 0.05, 0.1, 0.2, 0.5, 0.7 and 1 mg / ml. B. Sample preparation 1. Develop variants of A. niger in 96W filter MTP's (Millipore, # MAGVS2210), containing 200 μl of minimal medium + maltose (see above) per well for 7 days at 33 ° C, on an orbital shaker (225 rpm) with 80 to 90% humidity. 2. After the growth incubation, the cultures are filtered using a vacuum pipe, the filtrate (supernatant) is collected in a fresh 96W flat MTP (Greiner, # 655101), stored at 4 ° C. C. Severe incubation at elevated temperatures 1. Dilute 60 μl of the supernatant (sup) per well with 60 μl of 100 mM sodium acetate pH 4.85 (1: 1). (Optional: When the remaining supernatant needs to be checked for residual sugars, add 190 μl of 100 mM sodium acetate pH 4.8 to 10 μl of the supernatant per well, and transfer 20 μl of this diluted supernatant to 150 μl of the reagents PAHBAH for perform the sugar reduction assay of PAHBAH (see E). 2. Transfer 20 μl of diluted supernatant to a fresh 96W flat MTP and store at 4 ° C (for initial activity). 3. Incubate the remaining diluted supernatant (approximately 100 μl) for 1 hour at 61 ° C (or 65 ° C) (for residual activity). 4. Cool on ice for 10 minutes. D. Incubation of PASC; small scale conversion assay (SSC) 1. Fresh MTP's are prepared in flat: 180 μl / well of 0.5% PASC solution, well stirred, in 50 mM sodium acetate, pH 4.85. 2a. In a plate designed to measure the residual activity, 20 μl of treated diluted supernatant (sup) is transferred (with mixing up and down) per well after pre-incubation to the PASC-MTP's. 2b. On a second plate designed to measure the initial activity, transfer (with up and down mixing) per well, 180 μl of the PASC solution to 20 μl of the untreated, stored, supernatant diluted. 3. The sup-PASC MTPs are sealed and incubated for 2 hours at 50 ° C, shaken at 900 rpm. 4. Cool on ice for 10 minutes. 5. Transfer the sup-PASC mixture to a fresh MTP filter, filter in a vacuum tube and collect the filtered out . E. PAHBAH Reducer Sugar Assay 1. Prepare a fresh 96W flat MTP with 150 μl of the PAHBAH reagents per well. 2. Transfer 20 μl of the sup-PASC filtrate to the PAHBAH (mixing up and down). 3. A calibration line is placed in the column 1 in the first MTP; 20 μl of the cellobiose reserve solutions (see A4, above). 4. Incubate for 1 hour at 69 ° C, 900 rpm, cool to room temperature and centrifuge at 2000 rpm for 2 minutes. 5. The end point of OD410 is measured in the reader of MTP directly on the SpectraMax spectrometer (Spectra, Sunnyvale, CA, U.S.A.). F. Data processing (Spad-it) 1. From the readings on the cellobiose dilution wells, a cellobiose calibration line is plotted in mg / ml cellobiose versus OD410. 2. The calibration curve and the sample well readings are used to calculate, in mg / ml of cellobiose, the initial and residual values for each sup. 3. Percent residual activity is calculated Measurements of Residual Activity for the CBH2 Variants from Mutagenesis Directed to Si tio Table 14: Residual Activity of Wild Type Celsa and Variants The above table shows the percentage of remaining residual activity for the mutants directed to a single site (SDM, Example 3) after 1 hour of severe incubation at 61 ° C or at 65 ° C. The residual activities of the WT clones are shown in each subgroup of the data (there was a WT reference on each plate). The average value for the WT was 21.4% and 6.6% at 61 ° C and 65 ° C, respectively. It is clear that each mutant with a residual activity greater than WT is a molecule with improved thermostability under the selection conditions.

Residual Activity Measurements for Combinatorial Mutants of CBH2 The two columns of results in Tables 15 and 16 show the percentage residual activities after incubation at two different temperatures for the variants produced as in Example 4. Two tables (Tables 15 and 16) are presented immediately because the values were generated in two independent experiments. It is clear that each mutant with a residual activity greater than WT is a molecule with improved thermostability under the selection conditions. Table 15: Residual Activities for the Wild Type and the CelßA Variants Unless indicated otherwise, average residual residual activities were calculated from 4 determinations. Where "(8)" is indicated, 8 determinations were made. Standard deviation = standard deviation calculated for the determinations. Table 16: Residual activities for the wild type and the CelßA variants The average residual residual activities were calculated from the number of determinations indicated in parentheses. Standard deviation = standard deviation calculated for the decays. Residual Activity Measurements for CBH2 variants from Mutagenesis Regional Table 17 shows the% residual activity after incubation at 61 ° C for the variants produced as in Example 5.

Table 17: Residual Activities for the Wild Type and Variants of Cel6 Unless stated otherwise, each percentage residual activity number is the average of three determinations. Where "(32)" is indicated, 32 determinations were made. Standard deviation = standard deviation calculated for the determinations. It is obvious that each mutant with a residual activity greater than WT and / or S316P is a molecule with improved thermostability under the selection conditions.

EXAMPLE 9 Thermostability of CBH2 variants by Tm measurements CBH2 cellulase mutants were cloned, expressed and purified as described above (Example 7). The thermal denaturation data was collected on a microcalorimeter VP-DSC from Microcal (Nothampton, Mas sachuse 11 s, US). Buffer conditions were 50 mM Bis Tris Propane / 50 mM ammonium acetate / glacial acetic acid at pH 5.5 or pH 5.0, as indicated. The protein concentrations were approximately 0.25 mg / ml. Three thermal scans were carried out from 25 ° C to 80 ° C at a scanning speed of 90 ° C / hour. The first exploration showed thermal denaturation of the CBH2 and was used to determine the apparent intermediate point of the thermal denaturation, Tm: the instrumental software generates a curve of Cp (cal / ° C) versus Temperature (° C) and the Tm was determined manually from this curve. The thermal denaturation was irreversible in all cases, as shown by the absence of thermal denaturation in the second and third thermal explorations.

Table 18: Tm's by DSC at pH 5.5 glycosylated (rCBH2 wild type) Tm. "Tm G" = Tm measured on the purified recombinant protein. "Tm DG" = Tm measured on the recombinant protein deglycosylated with EndoH before being purified. Table 19: Tm's by DSC at pH 5.0 All variant data are referred to the glycosylated FCA500 (rCBH2 wild type) Tm. "Tm G" = Tm measure on the purified recombinant protein. "Tm DG" = Tm measured on the recombinant protein deglycosylated with EndoH before being purified. Mutations introduced into the CBH2 cellulase mutants affected the thermal stability of the CBH2 mutant cellulase compared to the wild type. The deglycosylated proteins used in this example and in the following Examples were prepared using procedures well known in the art for elimination of N-linked glycans (see, for example, Biochem J. (2001) 358: 423-430). See also Tai, T., et al. J. Biol. Chem. (1975) 250, 8569. EXAMPLE 10 Specific Activity of the CBH2 variant on PASC This example examines the specific performance on the puffed cellulose with phosphoric acid of the CBH2 variants compared to H. j ecorina CBH2. of wild type that had been cloned into A. Niger. Cellulose swollen with phosphoric acid (PASC) - PASC was prepared from Avicel according to the method described in Walseth (1971) Tappi 35: 228 (1971) and Wood Biochem J. 121: 353 (1971). This material was diluted with buffer and water to obtain a 1% w / v mixture such that the final concentration of sodium acetate was 50 mM, pH 5.0.

The relative specific performance of these variants on cellulosic substrates was determined by techniques known in the art. See, for example, Baker et al, Appl Biochem Biotechnol 1998 Spring; 70-72: 395-403. A standard cellulosic conversion assay was used in the experiments. See Baker supra. In this assay, the buffered enzyme and substrate were placed in containers and incubated at a temperature over time. The reaction was quenched with sufficient 100 mM glycine, pH 11.0 to bring the pH of the reaction mixture to at least pH 10. Once the reaction was quenched, an aliquot of the reaction mixture was filtered through a membrane. 0.2 micrometer to remove the solids. The filtered solution was then evaluated for the soluble sugars by HPLC according to the methods described in Baker et al., Appl. Biochem. Biotechnol. 70-72: 395-403 (1998). The relative specific activity of these variants was determined with 1% PASC in 50 mM sodium acetate pH 5.0 at 53 ° C with shaking at 1400 rpm for 3.5 hours. The enzymes were dosed at 0.75, 1.5 and 3 mg / g of cellulose. The protein concentration was determined by optical density (OD) 280 as in Leach and Scheraga 1960 (J. Am. Chem. Soc. 82: 4790-4792). The variants that were compared were FCA500.3, FCA523, FCA536, and FCA540-550.

For simplicity, Figure 8 shows only FCA540, FCA542, FCA545, FCA547, FCA549, and FCA550. All other variant samples had specific activities linked by the lines defined by the results of FCA542 and FCA545. Several of the new variant CBH2 from the selection of stability to temperature are as active as the wild type. To compare the numbers of the dose-dependent data, above (as shown in Figure 8), the proportion of total (average) sugar produced by one variant and the total (average) sugar produced by FCA500.3 (wild type) at the same dose, were averaged. These proportions, presented in Figure 9, are all very similar except for FCA547, FCA549 and FCA550, which are much less active. The error bars are the simple standard deviation of the average of the proportions. A ratio of 1 could indicate that the variant has activity similar to the wild type in this assay. All stabilized variants retained activity on this substrate. EXAMPLE 11 Specific Activity of the CBH2 variants on PCS This example compares the specific activity on pretreated corn cobs of the CBH2 variants compared to wild type H. j ecorina CBH2, which had been cloned inside A. Niger. Pretreated corn cob (PCS) - The corn cob was pretreated with 2% w / w sulfuric acid as described in Schell, D. et al., J. Appl. Biochem. Biotechnol. 105: 69-86 (2003) and followed by multiple washes with deionized water to obtain a paste having a pH of .5. Sodium acetate buffer (pH 5.0) was then added (up to a final concentration of 50 mM sodium acetate) and, if necessary, this mixture was then titrated to pH 5.0 using 1N sodium hydroxide. The concentration of cellulose in the reaction mixture was approximately 7%. The specific performance of the CBH2 was tested using PCS at 53 ° C with 700 rpm for 20 hours. Three different doses of the variants of CBH2, 0.75, 1.5 and 2.5 mg / g of cellulose (in the PCS) were added to 8.5 mg of broth of the cellulose strain deleted in CBH2 / g of cellulose. (For a discussion of the deletion of the CBH2 gene in Hypocrea j ecorina (also referred to as Trichoderma reesei) see U.S. Patent Nos. 5,861, 271 and 5,650,322). The results are shown in Figure 10. The baseline activity of the suppressed strain in CBH2 (without added CBH2) is shown. The CBH2 variant has similar activity to wild-type CBH2 in a whole cellulase reconstituted on PCS. This shows that the wild type, when it is added back to a suppressed strain gives some activity for above the suppressed strain. The variant achieves approximately the same activity under similar conditions. Similar runs were run for the other variants as described above. The total sugar values for the duplicates were averaged at each dose and then this value was divided by the average of the corresponding duplicates for FCA500.3 (wild type). These proportions, presented in Figure 11, are all very similar except for FCA547, FCA549 and FCA550 much less active. The error bars are the simple standard deviation of the proportions at different doses. A ratio of 1 could indicate that the variant has activity similar to the wild type in this assay. All stabilized variants retained activity on this substrate. EXAMPLE 12 Specific Activity of the Variant of CBH2 at Various Temperatures This example demonstrates how long each of the enzymes (variants and stabilized wild type) remained active at various temperatures. The assays described in Example 10 were used in this Example but modified as shown below. The total sugar produced by CBH2 (0.5 mg / g of cellulose) in 1% PACS at 53, 65 and 75 ° C with agitation at 300 RPM in several incubation times was used to determine how long each of these enzymes remained active (variants and stabilized wild type) at these temperatures. At 53 ° C, the variant possessed approximately the same activity as the wild-type enzyme over time (see Figure 12). Due to the stability of the enzymes at 53 ° C the half-lives of the enzymes could not be determined from the data. At 65 ° C, the total sugar produced by FCA543 and FAC500 shows that FCA543 is active for a longer period of time than FCA500 (Figure 13). The half-life of the variant was determined to be about 24 hours while the half-life of the wild-type was about 4 hours. However, both enzymes start to fail within the 72 hour incubation time. At 75 ° C, FCA543 produces more sugar than FCA500 in the first hour (see Figure 14). EXAMPLE 13 Specific Activity of the CBH2 Variant with Other Cellulases This example demonstrates the use of the (for example, stabilized) variant of CBH2 in the conversion of biomass in combination with other cellulases.

A mixture of three enzymes with the nucleus of Acidothermus cel l ulyti cus El (see WO 05/093050) plus CBHl and 2 wild type (FCA301 and FCA500, respectively) or CBHl and 2 stabilized (FCA469 and FCA543, respectively) was tested to 5 mg / g of cellulose and 10 mg / g of cellulose and to 38, 53 and 65 ° C in the standard conversion assay using PCS as a substrate (see Example 11). Samples were turned off at one, two and five days. The variants of CBHl are described in U.S. Patent Publication No. 20050054039, incorporated by reference herein. The enzyme Aci do th erm u s ce l l u l or lyt i c u s is described in U.S. Patent No. 5,712,142. Reference is also made to the following patent documents WO 91/05039; WO 93/15186; U.S. Patent No. 5,275,944; WO 96/02551; U.S. Patent No. 5,536,655 and WO 00/70031. Reference is also made to GenBank U33212. The results show that the specific functioning of the mixture of variants is approximately the same as that of the wild-type mixture at 38 ° C. (See Figure 15). A similar pattern is observed for operation at 53 ° C (data not revealed) . The mixture of stabilized variants shows a significant increase in specific performance on the wild-type mixture at 65 ° C (see Figure 16). Using the same standard conversion assay as described in Example 11, the specific operation of the mixture of stabilized variants was tested at 56, 59 and 62 ° C at 5 and 10 mg / g cellulose, and the samples were turned off 24, 48 and 120 hours. At all these three times, 56 ° C was better than the highest temperatures. See Figure 17. The optimum temperature is below 59 ° C at all times tested. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with the specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Rather, various modifications of the modes described to carry out the invention are obvious to those skilled in biology. molecular or related fields, are intended to be within the scope of the claims. REFERENCES 1. Sheehan and Himmel Biotechnology Progress 15, p. 817-827 (1999) 2. Matti Linko Proceedings of the Second TRICEL Symposium on Trichoderma reesei Cellulases and Other Hydrolases p. 9-11 (1993) 3. Tuula T. Teeri Trends in Biotechnology 15, p. 160- 167 (1997) 4. TT. Teeri et al. Spec. Publ.-R. Soc. Chem., 246 (Recent Advances in Carbohydrate Bioengineering), pp 302-308. (1999) 5. PDB reference 1QK2 (Cel6A = CBH2) J.-Y. Zou, GJ. Kleywegt, J. Stahlberg, H. Drigues, W. Nerinckx, M. Claeyssens, A. Koivula, T.T. Teeri ,. T.A Jones, Structure (LONDON), V. 7 p. 1035 (1999) 6. PDB reference 2BVW Structural changes of the active site tunnel of Humicola insolens cel lobiohydrolase, Cel6A, upon oligosacchar ide binding., Varrot A, Schulein M, Davies GJ, Biochemistry 1999 Jul 13; 38 (28): 888-91. 7. PDB reference 1DYS Structure and function of Humicola insolens family 6 cellulases: structure of the endoglucanase, CelßB, at 1.6 A resolution., Davies GJ, Brzozowski AM, Dauter M, Varrot A, Schulein M, Biochem J 2000 May 15; 348 Pt 1: 201-7. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Claims (1)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. A variant of cellulase CBH2, characterized in that the variant comprises a substitution or deletion in a position corresponding to one or more residues V94, P98, G118, M120, M134, T142, L144, M145, T148, T154, L179, Q204, V206, S210, 1212, T214, L215, G231, T232, V250, Q276, N285, S291, G308, T312, S316, V323, N325, 1333, G334, S343, T349, G360, S380, A381, S386, F411, S413, A416, Q426 and / or A429 in CBH2 of Hypocrea j ecorina (SEQ ID NO: 2). 2. The cellulase variant CBH2 according to claim 1, characterized in that the variant comprises a substitution at a position corresponding to one or more residues V94E, P98L, G118P, M120L, M134 (G / LV /), T142V, L144 ( G / R / S), M145L, T148Y, T154A, L179A, Q204E, V206L, S210 (L / R), 1212V, T214 (M / Y), L215I, G231N, T232V, V250I, Q276L, N285Q, S291G, G308A , T312S, S316P, V323 (L / N / Y), N325D, I333L, G334A, S343P, T349L, G360R, S380T, A381T, S386P, F411Y, S413Y, A416G, Q426E and / or A429T in Hypocrea's CBH2 j ecorina ( SEQ ID NO: 2). 3. The cellulase variant CBH2, characterized in that the variant comprises a substitution in a position corresponding to one or more of the residues V94, P98, G118, M120, M134, T142, L144, M145, T148, T154, L179, Q204, V206, S210, 1212, T214, L215, G231, T232, V250, Q276, N285, S291, G308, T312, S316, V323, N325, 1333, G334, S343, T349, G360, S380, A381, S386, F411, S413, A416, Q426 and / or A429 in Hypocrea j ecorina CBH2 (SEQ ID NO: 2). 4. The cellulase variant CBH2, characterized in that the variant comprises a substitution at a position corresponding to a residue selected from the group consisting of V94E, P98L, G118P, M120L, M134L, T142V, L144R, M145L, T148Y, T154A, L179A, Q204E, V206L, S210R, 1212V, T214Y, L215I, G231N, T232V, V250I, Q276L, N285Q, S291G, G308A, T312S, S316P, V323 (N / Y), N325D, I333L, G334A, S343P, T349L, G360R, S380T , A381T, S386P, F411Y, S413Y, A416G, A429T and Q426E in CBH2 of Hypocrea jecorina (SEQ ID NO: 2). 5. The CBH2 cellulase variant, characterized in that the CBH2 variant consists essentially of the mutations selected from the group consisting of: i. I212V / S316P / F411Y; ii. M134G / L144G / S316P; iii. M134L / L144R / S316P; iv. M134L / L144S / S316P; v. M13 V / V206L / I212V / T312S / S316P / F411Y / S413Y; saw. P98L / M134L / L144R / S210L / T214Y / S316P / V323Y / S413Y vii. P98L / M134L / L144R / S210R / T214Y / S316P / V323Y / S413Y viií. P98L / M134L / L144R / S316P / S413Y ix. P98L / M134L / L144R / S316P / V323Y / S413Y x. P98L / M134L / L144R / V206L / S210R / T214Y / S316P / S413Y xi. P98L / M134L / L144R / V206L / S210R / T214Y / S316P / V323Y / S413Y xii. P98L / M134V / I212V / S316P / S413Y xiii. P98L / M134V / I212V / T312S / S316P / S413Y xiv. P98L / M134V / S316P xv. P98L / M134V / S316P / S413Y xvi. P98L / M134V / S316P / V323Y / S413Y xvii. P98L / M134V / T154A / I212V / S316P / F411Y / S413Y xviii. P98L / M134V / T154A / I212V / S316P / S413Y xix. P98L / M134V / T154A / I212V / T312S / S316P / S413Y xx. P98L / M134V / T154A / T312S xxi. P98L / M134V / T154A / V206L / I212V / S316P / F411Y / S413Y xxii. P98L / M134V / T154A / V206L / S316P xxiii. P98L / M134V / V206L / F411Y xxiv. P98L / M134V / V206L / I212V / S316P / F411Y xxv. P98L / M134V / V206L / I212V / T312S / S316P / S413Y xxvi. P98L / M134V / 206L / S210R / T214Y / S316P / S413Y xxvii. P98L / M134V / 206L / S210R / T214Y / S316P / V323Y / S413Y xxviii. P98L / M134V / V206L / S316P / S413Y xxix. P98L / T154A / I212V / F411Y xxx. P98L / V206L / I212V / T312S / S316P / F411Y / S413Y xxxi. S316P / S413Y xxxii. S316P / V323L xxxiii. S316P / V323Y xxxiv. V206L / I212V / S316P xxxv. V206L / I212V / T312S / S316P xxxvi. V206L / I212V / T312S / S316P / F411Y / S413Y xxxvii. V206L / I212V / T312S / S316P / S413Y xxxviii. V206L / I212V / T312S / S316P / S413Y xxxix. V206L / S210L / T214M / S316P xl. V206L / S210R / S316P xli. V206L / 210R / T214Y / S316P; and xlii. V206L / S316P; in CBH2 of Hypocrea j ecorina (SEQ ID NO: 2). 6. A nucleic acid, characterized in that it encodes a variant of CBH2 according to claim 1. 7. A nucleic acid, characterized in that it encodes a variant of CBH2 according to claim 4. 8. A nucleic acid, characterized in that encodes a variant of CBH2 according to claim 5. 9. A vector, characterized in that it comprises a nucleic acid encoding a variant of CBH2 according to claim 6. 10. A vector, characterized in that it comprises a nucleic acid that codes for a CBH2 variant of according to claim 7. 11. A vector, characterized in that it comprises a nucleic acid encoding a variant CBH2 according to claim 8. 12. A host cell, characterized in that it is transformed with the vector according to claim 9. 13. A host cell, characterized in that it is transformed with the vector according to claim 10. 14. A host cell, characterized in that it is transformed with the vector according to claim 11. 15. A method for producing a variant of CBH2, characterized in that it comprises the steps of: (a) culturing a host cell according to claim 12, in a suitable culture medium, under conditions suitable to produce the variant CBH2; (b) obtain the CBH2 variant produced. 16. A method for producing a variant of CBH2, characterized in that it comprises the steps of: (a) culturing a host cell according to claim 13, in a suitable culture medium, under conditions suitable to produce the variant CBH2; (b) obtain the CBH2 variant produced. 17. A method for producing a variant of CBH2, characterized in that it comprises the steps of: (a) cultivating a host cell according to claim 14, in a suitable culture medium, under conditions suitable to produce the variant CBH2; (b) obtain the CBH2 variant produced. 18. A detergent composition, characterized in that it comprises a surfactant and a variant of CBH2, wherein the variant of CBH2 comprises a variant of CBH2 according to claim 1. 19. The detergent according to claim 18, characterized in that the detergent It is a laundry detergent. 20. The detergent according to claim 18, characterized in that the detergent is a cleaning agent. 21. A food additive, characterized in that it comprises a variant of CBH2 according to claim 1. 22. A method of treating wood pulp, characterized in that it comprises contacting the wood pulp with a variant of CBH2 in accordance with with the claim

1. 23. A method for converting biomass to sugars, characterized in that it comprises contacting the biomass with a variant of CBH2 according to claim 1.